Antibodies binding to an intracellular PRL-1 or PRL-3 polypeptide

ABSTRACT

We provide an antibody capable of binding to an intracellular PRL-1 or PRL-3 polypeptide, in which the antibody is capable of binding to an epitope bound by antibody 269, antibody 223 or antibody 318. Such anti-PRL antibodies may be capable of binding to intracellular PRL-1 or PRL-3. They may be suitable for use as therapies against cancer or metastasis thereof, or in clinical diagnosis to identify PRL-3 or PRL-1 positive patients.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Phase Entry Application ofInternational Application No. PCT/SG2008/000161 filed May 3, 2008, whichdesignated the U.S., and which claims the benefit of priority under 35U.S.C. §119(e) of U.S. Provisional No. 60/924,201 filed May 3, 2007, andU.S. Provisional No. 61/064,229 filed Feb. 22, 2008, the contents ofwhich are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 25, 2013, isnamed 629156US.txt and is 16,452 bytes in size.

FIELD

The present invention relates to the fields of medicine, cell biology,molecular biology and biochemistry. This invention also relates to thefield of medicine.

In particular, it relates to treatment and diagnosis of diseases, inparticular cancer, as well as compositions for such use.

BACKGROUND

Cancer is a serious health problem across the world. It is estimatedthat 7.6 million people in the world died of cancer in 2007. In the UKfor example, cancer is responsible for 126,000 deaths per year. One infour people die from cancer.

Known treatments for cancer include surgery, chemotherapy andradiotherapy. Many cancers can be cured if detected early enough.

100 years ago, the concept of antibodies as “magic bullets” was proposedby the German chemist Paul Ehrlich. Antibodies are capable ofrecognising and binding to their antigens in a specific manner and aretherefore ideal agents for recognizing and destroying malignant cellsvia the immune system. For this reason, they constitute the most rapidlygrowing class of human therapeutics for cancer.

A number of potential cancer or tumour markers and cancer antigens havebeen identified in the literature and antibody therapies have beendeveloped against some of them.

For example, the well-known cancer therapy Herceptin (Trastuzumab) is amonoclonal antibody that can kill HER2-positive cancer cells. Herceptinbinds to the HER2 (human epidermal growth factor receptor 2) antigen onthe cancer cell. Likewise, Bevacizumab (Avastin™) is a monoclonalantibody targeted against vascular endothelial growth factor (VEGF), oneof the growth factors implicated in the formation of new blood vessels.By inhibiting angiogenesis, Bevacizumab prevents tumour cells fromreceiving a constant supply of blood to receive the oxygen and nutrientsthe tumour needs to survive.

However, the applicability of antibody therapeutics for differentcancers is not universal. One of the limitations that has prevented thegeneral use of antibody therapeutics is the large size of antibodymolecules and their consequent inability to cross the plasma or cellmembrane. In the absence of modification, antibodies (includingmonoclonal antibodies) are only generally suitable for targeting cancerantigens located at the surface or exterior of host cells¹⁴⁻¹⁵. In theexamples above, HER2 receptor is located on the cell surface and ishence accessible for antibody binding by Herceptin. Likewise, VEGF issecreted into the bloodstream and is able to be bound by Bevacizumab.

PRLs are intracellular C-terminally prenylated proteins. Mutant forms ofPRLs that lack the prenylation signal are often localized innuclei¹⁶⁻¹⁷. The localization of PRL-1 and PRL-3 to the inner leaflet ofthe plasma membrane and early endosomes was revealed by EM immunogoldlabeling¹⁸. Over-expression of PRL-3 and PRL-1 has been shown to beassociated with a variety of human cancers^(3-12,19). PRL-1 and PRL-3are known to be associated with tumour metastasis. It is known that mostcancer patients die from metastases and not from their primary disease.

There is an urgent need for effective ways of preventing cancermetastasis. Antibodies have not hitherto been used for targetingintracellular antigens or cancer markers because of the inability of theantibodies to cross the cell membrane and the consequent inaccessibilityof the antigen.

SUMMARY

We have now demonstrated that antibodies against PRL-1 and PRL-3 cansurprisingly bind to their intracellular targets.

According to the expectation in the literature, targeting intracellularPRLs with antibodies to ablate cancer cells and cancer metastasis hasnever been previously thought to be possible because of theirintracellular location. We have shown that this is not the case, andprovide for anti-PRL-1 and anti-PRL-3 antibodies as cancer therapies,particularly therapies for cancer metastasis.

Li et al (2005) described the generation of PRL-1 and PRL-3 specificmonoclonal antibodies. However, the sequences of these antibodies andthe sequences of the variable regions of these antibodies have not beenpublished. Furthermore, the hybridomas producing these antibodies havenot been and are not so far publicly accessible. Accordingly, theantibodies described in Li et al (2005) have not so far been madeavailable to the public. Furthermore, there is no suggestion that theantibodies may be used for therapy of cancer, in view of theintracellular location of PRL-1 and PRL-3.

We disclose the variable regions of two mouse monoclonal antibodiesagainst PRL-1 (269 and 223) and one mouse monoclonal antibody againstPRL-3 (318). We disclose epitopes bound by anti-PRL-1 antibody 269 andanti-PRL-3 antibody 318. We further disclose methods of producing theseantibodies as well as methods of making antibodies which have the sameor similar binding properties as these antibodies, each of which hashitherto not been possible.

According to a 1^(st) aspect of the present invention, we provide anantibody, capable of binding to an PRL-1 or PRL-3 polypeptide, in whichthe antibody is capable of binding to an epitope bound by antibody 269,antibody 223 or antibody 318, or a variant, homologue, derivative orfragment thereof.

The antibody may be capable of binding to an epitope on a PRL-1polypeptide bound by antibody 269. The antibody may comprise ananti-PRL1 antibody capable of binding to an epitope TYKNMR (SEQ ID NO:13) or TLNKFI (SEQ ID NO: 14), or both, or a variant, homologue,derivative or fragment thereof.

The antibody may be capable of binding to an epitope on a PRL-3polypeptide bound by antibody 223 or antibody 318. The antibody maycomprise an anti-PRL3 antibody capable of binding to an epitope KAKFYN(SEQ ID NO: 35) or HTHKTR (SEQ ID NO: 39), or both, or a variant,homologue, derivative or fragment thereof.

The antibody may comprise the variable region of monoclonal antibody 269(SEQ ID NO: 2, SEQ ID NO: 4), the variable region of monoclonal antibody223 (SEQ ID NO: 6, SEQ ID NO: 8) or the variable region of monoclonalantibody 318 (SEQ ID NO: 10, SEQ ID NO: 12).

There is provided, according to a 2^(nd) aspect of the presentinvention, an antibody comprising the variable region of monoclonalantibody 269 (SEQ ID NO: 2, SEQ ID NO: 4), or a variant, homologue,derivative or fragment thereof which is capable of binding PRL-1.

We provide, according to a 3^(rd) aspect of the present invention, anantibody comprising the variable region of monoclonal antibody 223 (SEQID NO: 6, SEQ ID NO: 8), or a variant, homologue, derivative or fragmentthereof which is capable of binding PRL-1.

We provide, according to a 4^(th) aspect of the present invention, anantibody comprising the variable region of monoclonal antibody 318 (SEQID NO: 10, SEQ ID NO: 12), or a variant, homologue, derivative orfragment thereof which is capable of binding PRL-3.

The antibody may be capable of binding to an intracellular PRL-1 orPRL-3 polypeptide. The antibody may be capable of crossing the plasmamembrane of a cell.

The antibody may be capable of binding to and inhibiting a biologicalactivity of PRL-1 or PRL-3, preferably protein tyrosine phosphatase(PTP) activity.

The antibody may be capable of preventing metastasis of a cancer,preferably colorectal cancer, ovarian cancer, breast cancer, livercancer, pancreatic cancer, prostate cancer, gastric cancer, lung cancer,penis cancer, cervical cancer, brain cancer, esophageal cancer, bladdercarcinoma, kidney renal cell carcinoma, ovary lymphoma and skinmelanoma. The cancer may comprise PRL-1 or PRL-3 expressing cancer.

The antibody may comprise a monoclonal antibody or a humanisedmonoclonal antibody.

As a 5^(th) aspect of the present invention, there is provided acombination comprising an anti-PRL-1 antibody and an anti-PRL-3antibody, each as described.

We provide, according to a 6^(th) aspect of the present invention, apharmaceutical composition comprising such an antibody or combination,together with a pharmaceutically acceptable excipient, diluent orcarrier.

The present invention, in a 7^(th) aspect, provides an antibody capableof binding to PRL-1 or PRL-3, which may comprise an antibody asdescribed, a combination as set out above or a pharmaceuticalcomposition as set out above for use in a method of treatment orprevention of cancer or metastasis thereof.

The method may comprise exposing a cancer cell to the antibody orcombination. The method may comprise administering a therapeuticallyeffective amount of the antibody, combination or composition to anindividual suffering or suspected of suffering from cancer. The cancermay comprise a metastatic cancer. The cancer may be a PRL-1 or PRL-3expressing cancer.

The cancer may comprise colorectal cancer, ovarian cancer, breastcancer, liver cancer, pancreatic cancer, prostate cancer, gastriccancer, lung cancer, penis cancer, cervical cancer, brain cancer,esophageal cancer, bladder carcinoma, kidney renal cell carcinoma, ovarylymphoma and skin melanoma.

The number of metastatic tumours in a treated individual may be reducedby at least 50% compared to an untreated individual. It may be reducedby at least 60%. It may be reduced by at least 70%. It may be reduced byat least 80%. The number of metastatic tumours in a treated individualmay be reduced by at least 90% compared to an untreated individual.

In a 8^(th) aspect of the present invention, there is provided anantibody as set out above, a combination as described or apharmaceutical composition as described for use in a method of diagnosisof a cancer or metastasis thereof.

According to an 9^(th) aspect of the present invention, we provide adiagnostic kit comprising such an antibody, such a combination or such apharmaceutical composition together with instructions for use in thediagnosis of a cancer or metastasis thereof.

We provide, according to a 10^(th) aspect of the invention, apolypeptide comprising a sequence selected from the group consisting of:SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 orSEQ ID NO: 12, or a variant, homologue, derivative or fragment thereofwhich is capable of binding PRL.

There is provided, in accordance with a 11^(th) aspect of the presentinvention, a nucleic acid comprising a sequence capable of encoding amolecule as set out above such as a sequence selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9 and SEQ ID NO: 11, or a variant, homologue, derivative orfragment thereof which is capable of encoding a polypeptide having PRLbinding activity.

As an 12^(th) aspect of the invention, we provide a cell comprising ortransformed with such a nucleic acid sequence or a descendent of such acell.

We provide, according to a 13^(th) aspect of the invention, there isprovided a method of producing an antibody as described, the methodcomprising providing such a cell and expressing the antibody from thecell.

According to a 14^(th) aspect of the present invention, we provide amethod of diagnosis of cancer, such as metastatic cancer, in anindividual, the method comprising exposing a biological sample from theindividual to an antibody as set out above and detecting binding betweenthe antibody and a PRL-1 or PRL-3 polypeptide.

There is provided, according to a 15^(th) aspect of the presentinvention, a method of treatment or prevention of cancer, such asmetastatic cancer, in an individual suffering or suspected to besuffering from cancer, the method comprising administering atherapeutically effective amount of an antibody as described, acombination as described or a composition as described, to theindividual.

The method may comprises a feature as set out in any of the aboveparagraphs.

According to a 16^(th) aspect of the present invention, we provide amethod of treatment or prevention of cancer, such as metastatic cancer,in an individual suffering or suspected to be suffering from cancer, themethod comprising diagnosing cancer in the individual by a method asdescribed and treating the individual by a method as described.

According to a 17^(th) aspect of the present invention, we providemethod of detecting a metastatic cell, the method comprising exposing acandidate cell to an antibody as described above and detectingexpression of PRL-1 or PRL-3 polypeptide by the cell.

According to a 18^(th) aspect of the present invention, we providemethod of producing an animal model for metastatic tumours, the methodcomprising: (a) administering a plurality of metastatic cancer cells,such as a PRL-1 or PRL-3 expressing cancer cells, into a first animal;(b) allowing the cells to develop into metastatic tumours in the firstanimal; (c) extracting a metastatic tumour from the first animal andderiving a cell line from the metastatic tumour; and (d) administering aplurality of cells of the cell line into a second animal.

According to an 19^(th) aspect of the present invention, we provide ananimal model obtainable by such a method.

According to a 20^(th) aspect of the present invention, we provide useof an animal model produced by such a method or as set out above as amodel for metastatic tumours.

According to a 21^(st) aspect of the present invention, we providemethod comprising the steps of providing an antibody as described andallowing the antibody to bind to a PRL-1 or PRL-3 polypeptide.

The antibody may be allowed to bind to a cell expressing a PRL-1polypeptide or a PRL-3 polypeptide. The PRL-1 may comprise anintracellular PRL-1 polypeptide. The PRL-3 polypeptide may comprise anintracellular PRL-3 polypeptide.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O′D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3. Each of these general texts is herein incorporated byreference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Seven-step animal model for rapid formation of aggressive lungtumor metastases 1. Generation of CHO stable pools with 50% of cellsexpressing EGFP-PRL-3 or EGFP-PRL-1 as described previously⁸. 2.Injection of 1×10⁶ of these cells via the tail vein into the circulationof nude mice. 3. Isolation of a single EGFP-PRL-3 or EGFP-PRL-1 lungtumor at 3-week post-injection. 4. Dissecting and mincing the tumor inculture dishes to generate homogeneous EGFP-PRL-expressing cell lines(AT-3 or AT-1). 5. Injection of 1×10⁶ AT-3 or AT-1 cells into thecirculation via the tail vein of nude mice. 6. Untreated groups: notreatment, PBS or unrelated mouse antibodies. 7. Mice injected with AT-3cells are treated with PRL-3 mAbs clones #223 or #318 in the form ofascitic fluid or purified IgG; while mice injected with AT-1 cells areeither treated with PBS or treated with PRL-1 mAb clone 269 asciticfluid.

FIG. 2. PRL-3 and PRL-1 mAbs specifically inhibit the formations oftheir respective metastatic lung tumors. A. 1×10⁶ AT3 cells (describedin FIG. 1) are injected into nude mice via the tail vein. Mice areeither untreated (a, n=10) or PBS-treated (b, n=10), or treated with twounrelated antibodies (c, n=5; d, n=5). PRL-3 mAb 223 is in the form ofpurified IgG (e) or ascitic fluid (g); PRL-3 mAb 318 is in the form ofpurified IgG (f) or ascitic fluid (h) administrated via the tail vein.The different antibodies are injected on days 3, 6, and 9post-inoculation of AT3 cells. Lungs are dissected out on day 15post-injection and photographed under fluorescence microscopy to showthe GPF-positive metastatic tumours. Images a, b, e, and f are lungsfrom female mice. Images c, d, g, and h are lungs from male mice. B. Thetotal numbers of tumour in A are quantified in the Y axis as the averageof tumour lesions from each group, while the X axis displays the variousgroups of mice with different treatments. The results from mice injectedwith AT-3 cells are shown in columns 1-7. The mice injected with AT-1cells are either treated with PBS or with mAb 269 against PRL-1 (columns8-9). n=numbers of mice in each group.

FIG. 3. PRL-1 mAb specifically blocks PRL-1 but not PRL-3 metastatictumours; while PRL-3 mAb specifically blocks PRL-3 but not PRL-1metastatic tumours. We injected one million cancer cells (AT-1 or AT-3)either EGFP-PRL-1 or -PRL-3 into each mouse via its tail vein on day 1.Mice are then divided into groups respectively receiving PBS, rabbit PRLantibodies, PRL-1 mAb or PRL-3 mAb via their tail veins on day 3, 6, and9 post-cancer cell injections. Lungs (panels: a-d) derived from micecarrying AT-1 cancer cells expressing EGFP-PRL-1 or lungs (e-h) derivedfrom mice carrying AT-3 cancer cells expressing EGFP-PRL-3 are dissectedout and photographed on day 15. PRL-1 and PRL-3 metastatic tumours inlungs are not blocked by mock-PBS treatment (a, e) but are botheffectively blocked by rabbit antibodies (b, f). PRL-1 mAb inhibits theformation of tumours in which PRL-1 is overexpressed (c) but not whenPRL-3 is overexpressed (g). Similarly, PRL-3 mAb blocks the formation ofmetastatic tumours in which PRL-3 is overexpressed (h) but not whenPRL-1 is overexpressed (d). Therefore, these individual PRL-mAbs arespecific in blocking formation of lung metastatic tumours of cellsexpressing the target antigen, respectively.

FIG. 4. PRL-3 mAbs effectively block metastatic tumour formation byA2780 PRL-3-positive cancer cells; but have no effect on metastatictumour formation by CT26 PRL-3 negative cancer cells. A. A2780 but notCT26 cells express endogenous PRL-3. Cell lysates prepared from CT26mouse colon cancer cell line and A2780 human ovarian cancer cell lineare analyzed by western blotting to detect PRL-3. GAPDH is used as aloading control. B. PRL-3 antibody treatment does not affect lung tumourformation of CT26 PRL-3 negative cells at 2-week post cancer cellinoculation. All major tissues are examined for tumour formation. Grossappearances the animals as well as the dissected lungs of treated mice(panel a) and of untreated mice (panel b) are imaged. Extensive tumourformation is observed in all lungs as indicated by black arrows. C.PRL-3 antibody treatment inhibits pathologic appearances and tumourformation of A2780 PRL-3 positive cells in experimental metastasis assayat 1-month post cancer cells inoculation. All major tissues are examinedfor tumour formation. Gross appearances of the animals and tumours(indicated by black arrows) dissected out from untreated animals areimaged. Tumours are not found in treated mice.

FIG. 5. Antibody uptake in AT-3 cancer cells and parental CHO cells arerevealed by indirect immunofluorescence. A, EGFP-PRL-3 is expressed inall non-permeabilized AT-3 cells as detected by EGFP (a, d). A few cellsare completely labeled with PRL-3 mAb¹⁹ (white arrows indicated in b, epanels); 40% of non-permeabilized cells are partially labeled in redwith the PRL-3 mAbs; the internalized antibody seems to be distributedin a polarized manner at cell edges (single-head arrows indicated in 0and some in membrane protrusions (double-head arrows indicated in 0;while the remainder of the cells remained unlabeled (c and f cells ingreen only). B. To-pro-3 iodide is used to stain the DNA of everyparental CHO cell in blue. Live cells (10-20%) capable of taking upmouse anti-GS28 are shown in green (a, b, and c). The majority (60-70%)of live cells that are serum-starved overnight are able to take up mouseanti-GS28 shown in green (d, e, and f). White arrows indicate some cells(perhaps in some stages of cell cycle) that are not able to up-takeantibody. Bars, 20 μm.

FIG. 6. The general phenomenon of antibody uptake in normal and cancercells is revealed by indirect immunofluorescence with double staining:A, mouse anti-GS28 (in Red) and rabbit anti-PTEN (in green) antibodiesare added to non-permeabilized MCF-10A normal cells. B. mouse anti-GS28(in red) and rabbit anti-p53 (in green) antibodies are added tonon-permeabilized MCF-7 cancer cells. C. mouse anti-GS28 (in red) andrabbit anti-p53 (in green) antibodies are added to non-permeabilized, 16h serum-starved MCF-7 cells. To-pro-3 iodide is used to stain DNA(blue). Bars, 20 μm.

FIG. 7. PRL-3 and PRL-1 are over-expressed in multiple human cancers. A.PRL-3 mAbs (clone 223 or 318) are used to examine the expression ofPRL-3 in various human cancer samples. Examples of PRL-3 over-expressionare shown in human colon (26/158 cases, a), breast (19/96 cases, b),esophagus (2/13 cases, c), lung (d, the smoke deposits at the upperright-hand side of the dotted line stained black), penis (e), and cervixcancers (f). The PRL-3 positive signals are shown in brown byimmunohistochemistry with 3,3′-diaminobenzidine chromogen (DAB).Magnification of all images is ×400. B. PRL-1 mAb (clone 269) is used toexamine the expression of PRL-1 in various human cancer samples.Examples of PRL-1 over-expression in human colon (8/128 cases, a, ×200magnification), brain (5/20 cases, b, ×200), and esophagus (1/13 cases,c, ×400) are shown.

FIG. 8. PRL-3 and PRL-1 chimeric mAbs specifically react only to theirrespective antigen. A. A model is shown to illustrate an outline of themajor steps for chimeric mAb construction B. By IF, PRL-3 chimeric mAb(#318) is tested on DLD-1 cells that overexpress EGFP-PRL-3 (a) andshowed that the PRL-3 chimeric mAb could recognize EGFP-PRL-3 in thesecells (b). Merged image is shown in c. Similarly, PRL-1 chimeric mAb(#269) is also tested on DLD-1 human colorectal cancer cells thatoverexpress EGFP-PRL-1 (d) and showed that the PRL-1 mAb could recognizeEGFP-PRL-1 in these cells (e). Merged image is shown in f. Bars: 20 μm.C. By western blot analysis, the PRL-3 chimeric mAb are assessed on 4cell lysates derived from DLD-1 cells that overexpress EGFP-PRL-3 (lane1), and CHO cells that overexpress myc-PRL-3 (lane 2), myc-PRL-1 (lane3) or myc-PRL-2 (lane 4). PRL-3 chimeric mAb only react with EGFP-PRL-3and myc-PRL-3 but not with myc-PRL-1 and myc-PRL-2. D. the PRL-1chimeric mAb is assessed on 3 GST-PRL proteins: GST-PRL-1 (lane 1),GST-PRL-2 (lane 2) and GST-PRL-3 (lane 3). PRL-1 chimeric mAb react onlywith GST-PRL-1 but not with GST-PRL-2 and GST-PRL-3.

FIG. 9. PRL-3 chimeric mAb dramatically inhibits the formation ofmetastatic tumours by A2780 cells and HCT116 that express endogenousPRL-3; but not DLD-1 cells that do not express endogenous PRL-3. A. Thetotal cell lysates are prepared from HCT116, A2780, DLD-1 cancer cells,and DLD-1 cells that overexpress EGFP-PRL-3. The endogenous PRL-3protein is detected in HCT116 and A2780 but not in DLD-1 cells. Theexogenous EGFP-PRL-3 is only detected in lane 4. For B, C, and D: Nudemice are injected with 1×10⁶ cancer cells via tail veins on day 1. Themice are either administrated with PRL-3 chimeric mAb (treated) or PBS(untreated). The time period of each experiment are determined and endedwhen untreated mice are too sick. The photos are taken at the ends ofeach experiment. B. treatment for HCT116 cells, treated mice at the leftand untreated mice at the right. C. treatment for A2780 cells, treatedmice at the left and untreated mice at the right. D. a. treatment forDLD-1 cells, treated mice at the left and untreated mice at the right.b. treatment for DLD-1 cells expressing EGFP-PRL-3, untreated mice atthe left and treated mice at the right.

FIG. 10. PRL-3 enhances lung metastatic tumour formation and cancer cellsurvival in the blood circulation. A. Lung sections from treated anduntreated mice are shown. Multiple micro-tumours (indicated withMicro-T) is seen under fluorescence microscope in lung section fromuntreated mice but less found in lung sections from treated mice. B.Blood samples obtained from tail veins from treated and untreated miceare smeared on the glass slides and examined for EGFP-PRL-3 cancer cellsunder fluorescence microscope. Arrows indicate the EGFP-PRL-3 positivecancer cells.

FIG. 11. PRL-3 chimeric antibody effectively inhibits the formation ofmetastatic tumours by B16F0 cells that express endogenous PRL-3; but notB16F10 cells that do not express endogenous PRL-3. A. Total cell lysatesare prepared from B16F0 and B16F10 cancer cells. The endogenous PRL-3protein is detected only in B16F0 but not in B16F10 cells. B. Nude miceare injected with 1×10⁶ B16F0 cells on day 1 followed by chimeric mAbtreatment. Treated mice at the left and untreated mice at the right areshown. Tumours are found in the adrenal, liver, bone, and abdomen inuntreated mice. PRL-3 mAb could eliminate the formations of tumour inmost tissues of treated mice. C. Nude mice are injected with 1×10⁶B16F10 cells on day 1. Treated mice at the top and untreated mice at thebottom are shown. Dozens of lung metastatic tumours are found both intreated and untreated mice.

FIG. 12. Anti-PRL3 antibody 318 binds to both intracellular andexternalised/secreted PRL-3 polypeptide. A. Intracellular PRL3, B. GAPDHcontrol, C. Externalised/secreted PRL-3 polypeptide.

FIG. 13. Epitope mapping of anti-PRL1 antibody 269 and anti-PRL3antibody 318. A. Peptides bound by anti-PRL1 antibody 269 as shown byWestern Blot. B. Peptides bound by anti-PRL3 antibody 318 as shown byWestern Blot.

DETAILED DESCRIPTION

Anti-PRL Antibodies

The Examples describe the generation and production of antibodiesagainst PRL proteins, i.e., anti-PRL antibodies. Such anti-PRLantibodies may be capable of binding PRL-1 or PRL-3, preferablyintracellular PRL-1 or PRL-3.

Both monoclonal antibodies and humanised monoclonal antibodies and theirproperties are described in detail in this document and the Examples.The antibodies include monoclonal antibody 269, capable of binding toPRL-1. They also include monoclonal antibody 223 and monoclonal antibody318, capable of binding to PRL-3. Humanised versions of each of theseantibodies are also disclosed.

For the avoidance of doubt, where a specific antibody designation isreferred to in this document, this should be taken to include areference to both the mouse monoclonal antibody (as secreted by ahybridoma), as well as to the humanised version of it, unless thecontext dictates otherwise. Thus, for example, where antibody 269 isreferred to, this includes both the monoclonal antibody 269 (i.e., themouse hybridoma secreted antibody designated 269), as well as ahumanised monoclonal antibody 269.

The specific antibodies described in this document may be produced by aperson skilled in the art from the information disclosed in thisdocument, and employing molecular biology techniques which we alsodescribe in detail.

For this purpose, we disclose the sequences of the variable regions ofmonoclonal antibody 269, monoclonal antibody 223 and monoclonal antibody318. We further disclose variants, homologues, fragments and derivativesof these variable regions. Using this sequence information, a skilledperson may produce antibodies comprising these variable regions or theirvariants, homologues, fragments and derivatives.

We further disclose the sequences of nucleic acid constructs forexpressing these monoclonal antibodies. The sequences of theseconstructs enable the production of monoclonal antibodies which haveidentical sequences to 269, 223 or 318. We further disclose variants,homologues, fragments and derivatives of 269, 223 and 318.

Finally, we disclose the sequences of constructs capable of expressingthe humanised monoclonal antibodies 269, 223 or 318. We describe methodsof expressing the antibodies of interest from cells transfected with theconstructs, as well as variants, homologues, fragments and derivativesof these humanised constructs.

Using such sequences and the expression methods, the skilled person mayreadily transfect relevant host cells and cause them to express thewhole monoclonal or humanised anti-PRL-1 and anti-PRL3 antibodies, orvariants, homologues, fragments and derivatives thereof.

We further provide for polypeptides in general having PRL bindingactivity. Such polypeptides include anti-PRL antibodies such asanti-PRL-1 antibodies and anti-PRL3 antibodies. The PRL-bindingpolypeptides may comprise one or more of the same or similar propertiesas the monoclonal antibodies 269, 223 and 318. The polypeptides will bereferred to for convenience generally as “anti-PRL antibodies”.

It is within the skills of a reader to construct binding molecules whichmay not be (or may not be described as) antibodies or immunoglobulinsbut which comprise anti-PRL binding activity as described here.Accordingly, and where the context allows the term “anti-PRL antibodies”should be taken to include any molecule so long as it is capable ofbinding PRL. Such molecules may include polypeptides, small molecules,as well as antibodies and immunoglobulins, and may be identified throughvarious means known in the art, for example by screening a suitablelibrary for PRL binding activity.

The anti-PRL antibodies (which include PRL binding molecules) maycomprise similar or identical properties may as the monoclonalantibodies 269, 223 and 318. Such similar or identical properties may inparticular include binding properties. The anti-PRL antibodies may ingeneral be capable of binding to PRL polypeptides, e.g., PRL-1 andPRL-3.

Thus, the term “anti-PRL antibody” will be taken to include monoclonalantibodies 269, 223 and 318 (as well as their humanised counterparts).Also included are polypeptides comprising the variable regions ofantibodies 269, 223 or 318 or variants, homologues, fragments andderivatives thereof. This term should also be taken to include referenceto variants, homologues, fragments and derivatives of the anti-PRLantibodies, as described below, where the context permits.

PRL1 and PRL3 Epitopes

The anti-PRL antibodies may have the same or similar bindingspecificity, binding affinity and/or binding affinity as 269, 223 or318. The anti-PRL antibodies may specifically bind to an epitope boundby antibody 269, an epitope bound by antibody 223 or an epitope bound byantibody 318.

Methods are known in the art to determine an epitope that is bound by aparticular antibody. Such epitope mapping methods are described forexample in Hanson et al., (2006). Respiratory Research, 7:126.Furthermore, a skilled person will be able to generate antibodies andscreen them for particular properties. A detailed description of such amethod is shown in Example 27. Accordingly, a skilled person willreadily be able to identify anti-PRL antibodies which bind to the sameepitopes as 269, 223 and 318.

Example 27 shows that anti-PRL1 antibody 269 binds epitopes TYKNMR (SEQID NO: 13) and TLNKFI (SEQ ID NO: 14). Accordingly, we provide ananti-PRL antibody such as an anti-PRL1 antibody capable of binding asequence TYKNMR (SEQ ID NO: 13). We further provide an anti-PRL antibodysuch as an anti-PRL1 antibody capable of binding a sequence TLNKFI (SEQID NO: 14). The anti-PRL antibody may be capable of binding bothsequences.

Furthermore, Example 27 shows that anti-PRL3 antibody binds epitopesKAKFYN (SEQ ID NO: 35) and HTHKTR (SEQ ID NO: 39). We therefore providean anti-PRL antibody such as an anti-PRL3 antibody capable of binding asequence KAKFYN (SEQ ID NO: 35). We further provide an anti-PRL antibodysuch as an anti-PRL3 antibody capable of binding a sequence HTHKTR (SEQID NO: 39). The anti-PRL antibody may be capable of binding bothsequences.

The anti-PRL antibodies may comprise the variable region of antibody269, or the variable region of antibody 223 or the variable region ofantibody 318, each of which is described in detail below. They maycomprise the same or different variable regions in a single antibodymolecule. They may comprise one variable region, or more than onevariable region. Accordingly, we provide the skilled person with theability to produce any number of antibodies which comprise the same orsimilar binding reactivity as antibody 269, 223 or 318.

Such antibodies may comprise the full or substantially completesequences of an antibody (i.e., heavy chain and light chain), or theymay comprise a fragment of a whole antibody (such as Fv, F(ab′) andF(ab′)₂ fragments or single chain antibodies (scFv)). The antibodies mayfurther comprise fusion proteins or synthetic proteins which comprisethe antigen-binding site of the antibody, as described in detail below.It will also be evident that such antibodies may be engineered fordesirable properties, such as lowered host reactivity, reducedrejection, etc.

The engineering could include “humanisation”, by which term we mean theinclusion of (or substitution with) one or more human residues orsequences in an antibody sequence such as a mouse antibody sequence.“Humanisation” in the context of this document includes “chimeric”antibodies, in which the antibody comprises discrete sections of mouseand human sequences, e.g., where one or both of the variable regionscomprise mouse sequences, and the remainder of the antibody molecule(such as the constant region) comprises human sequences. In suchchimeric antibodies, the whole of the variable regions of, for example,a mouse or rat antibody may be expressed along with human constantregions. This provides such a chimeric antibody with human effectorfunctions and also reduces immunogenicity (HAMA) caused by the murine Fcregion.

Generally, a “chimeric antibody” may refer to an antibody having eithera heavy and light chain encoded by a nucleotide sequence derived from amurine immunoglobulin gene and either a heavy and light chain encoded bya nucleotide sequence derived from a human immunoglobulin gene.

“Humanisation” also includes CDR grafted or reshaped antibodies. It thusincludes engineering at a more discrete level, e.g., antibodies in whichthe mouse variable region has been mutated to include human residues toreduce immunogenicity. In such an antibody, only the complimentaritydetermining regions from the rodent antibody V-regions may be combinedwith framework regions from human V-regions. Such antibodies should bemore human and less immunogenic than chimaeric antibodies.

The anti-PRL antibody may generally be capable of binding to PRLpolypeptide in a number of conditions.

In one embodiment, the binding environment comprises an intracellularcondition. That is to say, the anti-PRL antibody may be capable ofbinding to a PRL polypeptide in an intact or unpermeabilised cell. Suchan unpermeabilised cell may comprise a cell which has not been exposed,or not exposed substantially, to a permeabilisation agent such as adetergent (e.g., Triton X-100) or digitonin.

An anti-PRL-3 antibody as described here may be capable of binding toPRL-3 when it is inside the cell, within the cell membrane orencapsulated within the cell. Similarly, a PRL-1 polypeptide may bebound by an anti-PRL-1 antibody, as described generally in thisdocument, in the context of an environment that comprises the interiorof a cell. The anti-PRL-1 and anti-PRL3 antibodies may in particular becapable of binding to an intracellular PRL-1 or PRL-3 polypeptide. Theintracellular PRL polypeptide may be associated with one or a number ofcellular structures, for example, the inner leaflet of the cellmembrane, an organelle, a cytoskeletal structure, the nuclear membrane,etc. The PRL polypeptide may be located within the nucleus. In each ofthese cases, the anti-PRL antibody may be capable of binding to the PRLpolypeptide within the intracellular environment.

The anti-PRL antibody may be capable of binding to a PRL polypeptide inan intracellular environment in a number of ways. The anti-PRL antibodymay be capable of crossing the plasma membrane. It may be capable ofotherwise gaining access to a binding region of the PRL polypeptide, forexample by cellular uptake. It may be internalised or translocated orotherwise delivered into the cell by any means.

In another embodiment, the binding condition comprises an extracellularcondition. The anti-PRL antibody may therefore be capable of binding toits cognate PRL polypeptide in an extracellular environment.

The anti-PRL antibody may therefore be capable of binding to a PRLpolypeptide extracellularly. In other words, an anti-PRL-1 antibody asdescribed here may be capable of binding to PRL-1 when it is outside thecell. Similarly, a PRL-3 polypeptide may be bound by an anti-PRL-3antibody, as described generally in this document, in the context of anenvironment that is external to the interior of a cell. The anti-PRLantibody may be capable of binding to a secreted PRL-1 or PRL-3polypeptide, as the case may be. The PRL-1 or PRL-3 polypeptide maycomprise a circulating PRL-1 or PRL-3 polypeptide.

The anti-PRL antibody may be capable of binding to bind to external orexternalized PRL polypeptides. They may bind to secreted PRLpolypeptides in blood circulation.

The binding between the anti-PRL antibody and its target may be more orless strong or weak, transient, semi-permanent or permanent.

Binding of the anti-PRL antibody to the PRL polypeptide may take placewithin the cell. Such binding may inactivate, inhibit or lower anactivity of the PRL polypeptide. The binding may neutralise a PRLactivity. The activity may comprise any biological activity caused by orassociated with the PRL polypeptide. The activity may comprise bindingto another protein, for example a downstream protein or factor. Bindingof anti-PRL antibody to PRL polypeptide may inactivate, inhibit or loweran activity of a downstream protein or factor. The activity may comprisecommunication with other cells, for example cells such as metastaticcancer cells in circulation. Thus, the anti-PRL antibodies mayneutralise PRL polypeptides in blood circulation to preventPRL-phosphatases from binding with down-stream factors or from theircommunicating with other cells in circulation.

The activity may comprise a biochemical activity or a pathogenicactivity. The biochemical activity may comprise a catalytic activity.The catalytic activity may comprise phosphatase activity. The activitymay comprise growth regulating activity, cancer activity, carcinogenicactivity or metastatic activity.

The monoclonal antibodies 269, 223 and 318 may be used for treatment ofdisease in humans or other animals. We show in the Examples that suchanti-PRL antibodies have anti-cancer activity. Specifically, theExamples show that the anti-PRL antibodies are capable of preventingmetastatic spread of cancer tumours.

Example 9 describes the generation of PRL-over expressing tumours inmice and provides for an animal model for metastasis and cancer therapy.Examples 10 to 12 show that animals treated with anti-PRL antibodiesshow significantly fewer metastatic lung tumours compared to animals nottreated with anti-PRL antibodies. Specifically, the treated animals showabout 90% fewer tumours than the untreated animals. The anti-PRLantibodies are capable of binding to blocking the activity of PRLpolypeptide, despite its intracellular localisation. Our studiesrepresent the first examples of effectively (−90%) blocking metastasisby using monoclonal antibodies against their respective phosphatasesdespite their intracellular localization.

We also show that anti-PRL-3 monoclonal antibodies effectively block theformation of metastatic tumours by a human ovarian cancer cell lineA2780 that expresses endogenous PRL-3 protein.

Accordingly, we provide for the use of anti-PRL antibodies in thetreatment or prevention of disease, such as cancer. The cancer maycomprise a metastatic cancer. The anti-PRL antibodies may be used asdrugs or therapies to treat metastasis of a cancer, such as anestablished tumour. They may be used to prevent cancer or metastasisthereof.

The cancer which is treatable or preventable may include one which isassociated with expression or over-expression of a PRL protein. The PRLprotein may be a relevant member of the family. By this we mean that acancer which is associated with expression or over-expression of PRL-1may be treatable or preventable by anti-PRL-1 antibody such as 269, oran antibody having a similar or identical properties. Similarly, acancer which is associated with expression or over-expression of PRL-3may be treatable or preventable by anti-PRL-3 antibody such as 223 or318, or an antibody having a similar or identical properties.

The cancer may include any of a number of cancers, such as colorectalcancer, ovarian cancer, breast cancer, liver cancer, pancreatic cancer,prostate cancer, gastric cancer, lung cancer, penis cancer, cervicalcancer, brain cancer, esophageal cancer, bladder carcinoma, kidney renalcell carcinoma, ovary lymphoma and skin melanoma.

The treatment may comprise generally contacting a cancer cell, or a cellsuspected of being a cancer cell, with an anti-PRL antibody. The cellmay be exposed to an anti-PRL-1 antibody. It may or in addition beexposed to an anti-PRL-3 antibody. It may be exposed to both ananti-PRL-1 antibody and an anti-PRL-3 antibody. Where this is so, thecell may be exposed to both antibodies together, or individually insequence. The exposure may be repeated a number of times. Anycombination of anti-PRL-1 antibody and an anti-PRL-3 antibody inwhatever amount or relative amount, in whatever timing of exposure, maybe used.

We therefore provide for the use of combinations of anti-PRL-1antibodies and anti-PRL-3 antibodies, as described above, in thetreatment of disease such as cancer.

The cell may be an individual cell, or it may be in a cell mass, such asa cancer or tumour cell mass. The cell may be inside the body of anorganism. The organism may be one which is known to be suffering fromcancer, or it could be one in which cancer is suspected. The treatmentmay comprise administering the antibody or antibodies to the organism.As above, a single antibody may be administered, or a combination ofanti-PRL-1 antibody and an anti-PRL-3 antibody may be administered. Theadministration may be simultaneous or sequential, as described above.Thus, the treatment may comprise administering an anti-PRL-1 antibodysimultaneously or sequentially with an anti-PRL-3 antibody to theindividual.

The anti-PRL antibody may generally comprise any immunoglobulin capableof binding to a PRL molecule, as described in more detail below.

PRL-1

The following text is adapted from OMIM entry 601585.

PRL-1 is also known as Protein-Tyrosine Phosphatase, Type 4a, 1; PTP4A1,Phophatase of Regenerating Liver 1, PTP(CAAX1). The chromosomal locationof PRL-1 is at gene map locus 6q12.

Cellular processes involving growth, differentiation, and metabolism areoften regulated in part by protein phosphorylation anddephosphorylation. The protein tyrosine phosphatases (PTPs), whichhydrolyze the phosphate monoesters of tyrosine residues, all share acommon active site motif and are classified into 3 groups.

These include the receptor-like PTPs, the intracellular PTPs, and thedual-specificity PTPs, which can dephosphorylate at serine and threonineresidues as well as at tyrosines.

Diamond et al. 1994, Cell. Biol. 14: 3752-3762, described a PTP fromregenerating rat liver that is a member of a fourth class. The gene,which they designated Prl1, was one of many immediate-early genes andexpressed mainly in the nucleus. Over-expression of Prl1 in stablytransfected cells resulted in a transformed phenotype, which suggestedthat it may play some role in tumorigenesis.

By using an in vitro prenylation screen, Cates et al., 1996, CancerLett. 110: 49-55, isolated 2 human cDNAs encoding PRL1 homologs,designated PTP(CAAX1) and PTP(CAAX2) (PRL2; 601584), that arefarnesylated in vitro by mammalian farnesyl:protein transferase.Overexpression of these PTPs in epithelial cells caused a transformedphenotype in cultured cells and tumor growth in nude mice. The authorsconcluded that PTP(CAAX1) and PTP(CAAX2) represent a novel class ofisoprenylated, oncogenic PTPs.

Peng et al. 1998, J. Biol. Chem. 273: 17286-17295, reported that thehuman PTP(CAAX1) gene, or PRL1, is composed of 6 exons and contains 2promoters. The predicted mouse, rat, and human PRL1 proteins areidentical. Zeng et al. 1998, Biochem. Biophys. Res. Commun. 244:421-427, determined that the human PRL1 and PRL2 proteins share 87%amino acid sequence identity. By FISH, Peng et al. (1998) mapped thePRL1 gene to 6q12.

Where the term “PRL-1” is used, this should be taken to refer to anyPRL-1sequence, including a PRL-1 protein or a PRL-1 nucleic acid and anyfragment, variant homologue, derivative, variant thereof.

The properties and activities of PRL-1 are described in this document,for example, in the references.

[End of Text Adapted from OMIM]

Mouse and human PRL-1 proteins were described in detail in Zeng et al(1998), supra.

PRL-1 Sequences

The methods and compositions described here make use of PRL-1polypeptides, which are described in detail below. As used here, theterm “PRL-1” is intended to refer to a sequence set out in Table D1below.

TABLE D1 PRL-1 Sequences Unigene Description NM_003463.3 Homo sapiensprotein tyrosine phosphatase type IVA, member 1 (PTP4A1), mRNACR602427.1 full-length cDNA clone CS0DK012YJ03 of HeLa cells Cot 25-normalized of Homo sapiens (human) CR599216.1 full-length cDNA cloneCL0BB007ZF05 of Neuroblastoma of Homo sapiens (human) CR596545.1full-length cDNA clone CS0DK010YM06 of HeLa cells Cot 25- normalized ofHomo sapiens (human) CR749458.1 Homo sapiens mRNA; cDNA DKFZp779M0721(from clone DKFZp779M0721) BC045571.1 Homo sapiens protein tyrosinephosphatase type IVA, member 1, mRNA (cDNA clone MGC: 57320 IMAGE:4826233), complete cds AJ420505.1 Homo sapiens mRNA full length insertcDNA clone EUROIMAGE 2096405 AK312526.1 Homo sapiens cDNA, FLJ92892BC023975.2 Homo sapiens protein tyrosine phosphatase type IVA, member 1,mRNA (cDNA clone MGC: 1659 IMAGE: 2960001), complete cds U69701.1 Humanprotein tyrosine phosphatase hPRL-1N mRNA, partial cds U48296.1 Homosapiens protein tyrosine phosphatase PTPCAAX1 (hPTPCAAX1) mRNA, completecds AK081491.1 Mus musculus 16 days embryo head cDNA, RIKEN full-lengthenriched library, clone: C130021B01 product: protein tyrosinephosphatase 4a1, full insert sequence AK078120.1 Mus musculus adult malemedulla oblongata cDNA, RIKEN full-length enriched library, clone:6330521E18 product: protein tyrosine phosphatase 4a1, full insertsequence BC055039.1 Mus musculus protein tyrosine phosphatase 4a1, mRNA(cDNA clone MGC: 62623 IMAGE: 6396041), complete cds AK199907.1 Musmusculus cDNA, clone: Y1G0132L24, strand: minus, reference: ENSEMBL:Mouse-Transcript- ENST: ENSMUST00000061959, based on BLAT searchAK198788.1 Mus musculus cDNA, clone: Y1G0129D05, strand: plus,reference: ENSEMBL: Mouse-Transcript- ENST: ENSMUST00000061959, based onBLAT search AK192767.1 Mus musculus cDNA, clone: Y1G0109N22, strand:plus, reference: ENSEMBL: Mouse-Transcript- ENST: ENSMUST00000055216,based on BLAT search AK187266.1 Mus musculus cDNA, clone: Y0G0140O11,strand: plus, reference: ENSEMBL: Mouse-Transcript- ENST:ENSMUST00000061959, based on BLAT search BC086787.1 Mus musculus proteintyrosine phosphatase 4a1, mRNA (cDNA clone MGC: 102117 IMAGE: 30538771),complete cds BC094447.1 Mus musculus protein tyrosine phosphatase 4a1,mRNA (cDNA clone MGC: 102501 IMAGE: 3990529), complete cds AK150506.1Mus musculus bone marrow macrophage cDNA, RIKEN full-length enrichedlibrary, clone: I830008L20 product: protein tyrosine phosphatase 4a1,full insert sequence AK148288.1 Mus musculus B16 F10Y cells cDNA, RIKENfull-length enriched library, clone: G370079M23 product: proteintyrosine phosphatase 4a1, full insert sequence AK151533.1 Mus musculusbone marrow macrophage cDNA, RIKEN full-length enriched library, clone:I830031H07 product: protein tyrosine phosphatase 4a1, full insertsequence U84411.1 Mus musculus protein tyrosine phosphatase (PRL-1)mRNA, complete cds NM_011200.2 Mus musculus protein tyrosine phosphatase4a1 (Ptp4a1), mRNA BC003761.1 Mus musculus, protein tyrosine phosphatase4a1, clone IMAGE: 3590144, mRNA BC031734.1 Mus musculus, proteintyrosine phosphatase 4a1, clone IMAGE: 3157812, mRNA

A “PRL-1 polypeptide” may comprise or consist of a human PRL-1polypeptide, such as the sequence having Unigene accession numberNM_(—)003463.3.

Homologues variants and derivatives thereof of any, some or all of thesepolypeptides are also included. For example, PRL-1 may include UnigeneAccession Number U84411.1.

PRL-3

The following text is adapted from OMIM entry 606449.

PRL-3 is also known as Protein-Tyrosine Phosphatase, Type 4A, 3; PTP4A3.The chromosomal location of PRL-3 is at gene map locus 8q24.3.

In the heart, protein kinases regulate contractility, ion transport,metabolism, and gene expression. Phosphatases, in addition to their rolein dephosphorylation, are involved in cardiac hypertrophy anddysfunction.

By database searching and screening of a heart cDNA library, Matter etal. 2001, Biochem. Biophys. Res. Commun. 283: 1061-1068 identified acDNA encoding PTP4A3, which they termed PRL3. The deduced PRL3 proteinis 76% identical to PRL1 (PTP4A 1; 601585) and 96% identical to mousePrl3. Northern blot analysis revealed expression of an approximately2.3-kb PRL3 transcript predominantly in heart and skeletal muscle, withlower expression in pancreas. This expression pattern is distinct fromthe wider expression of PRL1 and PRL2 (PTP4A2; 601584). In situhybridization analysis localized PRL3 expression to cardiomyocytes. Trisglycine gel analysis showed that PRL3 is expressed as a 22-kD protein.Functional and mutation analyses indicated that phosphate cleavage isdependent on cys104 of PRL3. Overexpression of PRL3 resulted inincreased cell growth. Western blot analysis showed dephosphorylation ofp130cas (BCAR1; 602941) in response to angiotensin II (106150),suggesting a role for PRL3 in the modulation of intracellular calciumtransients induced by angiotensin II.

To gain insights into the molecular basis for metastasis, Saha et al.2001, Science 294: 1343-1346 compared the global gene expression profileof metastatic colorectal cancer with that of primary cancers, benigncolorectal tumors, and normal colorectal epithelium. PRL3 was expressedat high levels in each of 18 cancer metastases studied but at lowerlevels in nonmetastatic tumors and normal colorectal epithelium. In 3 of12 metastases examined, multiple copies of the PRL3 gene were foundwithin a small amplicon located at chromosome 8q24.3. Saha et al. (2001)concluded that the PRL3 gene is important for colorectal cancermetastasis.

Using the Stanford G3 radiation hybrid panel and database sequenceanalysis, Saha et al. (2001) mapped the PRL3 gene to surrounding marker145.20. The PRL3 gene is also tightly linked to marker SHGC-22154, whichis located at 8q24.3, approximately 3 Mb from the 8q telomere.

[End of Text Adapted from OMIM]

Mouse and human PRL-3 proteins were described in detail in Li et al(2005), Clin Cancer Res; 11:2195-204.

PRL-3 Sequences

The methods and compositions described here make use of PRL-3polypeptides, which are described in detail below. As used here, theterm “PRL-3” is intended to refer to a sequence set out in Table D2below.

Unigene Description AF041434.1 Homo sapiens potentially prenylatedprotein tyrosine phosphatase hPRL-3 mRNA, complete cds BT007303.1 Homosapiens protein tyrosine phosphatase type IVA, member 3 mRNA, completecds AK128380.1 Homo sapiens cDNA FLJ46523 fis, clone THYMU3034099NM_007079.2 Homo sapiens protein tyrosine phosphatase type IVA, member 3(PTP4A3), transcript variant 2, mRNA AY819648.1 Homo sapiens HCVp7-transregulated protein 2 mRNA, complete cds BC003105.1 Homo sapiensprotein tyrosine phosphatase type IVA, member 3, mRNA (cDNA clone MGC:1950 IMAGE: 3357244), complete cds NM_032611.1 Homo sapiens proteintyrosine phosphatase type IVA, member 3 (PTP4A3), transcript variant 1,mRNA AK311257.1 Homo sapiens cDNA, FLJ18299 U87168.1 Human proteintyrosine phosphatase homolog hPRL-R mRNA, partial cds AJ276554.1 Homosapiens mRNA for protein tyrosine phosphatase hPRL-3, short formBC066043.1 Mus musculus protein tyrosine phosphatase 4a3, mRNA (cDNAclone MGC: 90066 IMAGE: 6415021), complete cds AK190358.1 Mus musculuscDNA, clone: Y1G0102I03, strand: plus, reference: ENSEMBL:Mouse-Transcript- ENST: ENSMUST00000053232, based on BLAT searchCT010215.1 Mus musculus full open reading frame cDNA cloneRZPDo836H0950D for gene Ptp4a3, Protein tyrosine phosphatase 4a3;complete cds, incl. stopcodon AK147489.1 Mus musculus adult male brainUNDEFINED_CELL_LINE cDNA, RIKEN full-length enriched library, clone:M5C1053F14 product: protein tyrosine phosphatase 4a3, full insertsequence AK172192.1 Mus musculus activated spleen cDNA, RIKENfull-length enriched library, clone: F830102P03 product: proteintyrosine phosphatase 4a3, full insert sequence AK143702.1 Mus musculus 6days neonate spleen cDNA, RIKEN full-length enriched library, clone:F430011C20 product: protein tyrosine phosphatase 4a3, full insertsequence AF035645.1 Mus musculus potentially prenylated protein tyrosinephosphatase mPRL-3 (Prl3) mRNA, complete cds NM_008975.2 Mus musculusprotein tyrosine phosphatase 4a3 (Ptp4a3), mRNA AK014601.1 Mus musculus0 day neonate skin cDNA, RIKEN full-length enriched library, clone:4632430E19 product: protein tyrosine phosphatase 4a3, full insertsequence AK004562.1 Mus musculus adult male lung cDNA, RIKEN full-lengthenriched library, clone: 1200003F10 product: protein tyrosinephosphatase 4a3, full insert sequence AK003954.1 Mus musculus 18-dayembryo whole body cDNA, RIKEN full-length enriched library, clone:1110029E17 product: protein tyrosine phosphatase 4a3, full insertsequence BC027445.1 Mus musculus protein tyrosine phosphatase 4a3, mRNA(cDNA clone MGC: 36146 IMAGE: 4482106), complete cds

A “PRL-3 polypeptide” may comprise or consist of a human PRL-3polypeptide, such as the sequence having Unigene accession numberAF041434.1.

Homologues variants and derivatives thereof of any, some or all of thesepolypeptides are also included. For example, PRL-3 may include UnigeneAccession Number BC066043.1.

PRL-1 and PRL-3 Polypeptides

PRL-1 and PRL-3 polypeptides may be used for a variety of means, forexample, for production or screening of anti-PRL-1 and anti-PRL-3 agentssuch as specific PRL-1 and PRL-3 binding agents, in particular, anti-PRLantibodies. These are described in further detail below. The expressionof PRL-1 and PRL-3 polypeptides may be detected for diagnosis ordetection of cancer, in particular breast cancer.

A “polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.

“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, Proteins—Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al, “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

The term “polypeptide” includes the various synthetic peptide variationsknown in the art, such as a retroinverso D peptides. The peptide may bean antigenic determinant and/or a T-cell epitope. The peptide may beimmunogenic in vivo. The peptide may be capable of inducing neutralisingantibodies in vivo.

As applied to PRL-1 and PRL-3, the resultant amino acid sequence mayhave one or more activities, such as biological activities in commonwith a PRL-1 or PRL-3 polypeptide, for example a human PRL-1 or PRL-3polypeptide. For example, a PRL-1 or PRL-3 homologue may have aincreased expression level in cancer cells compared to normal breastcells. In particular, the term “homologue” covers identity with respectto structure and/or function providing the resultant amino acid sequencehas PRL-1 pr PRL-3 activity. With respect to sequence identity (i.e.similarity), there may be at least 70%, such as at least 75%, such as atleast 85%, such as at least 90% sequence identity. There may be at least95%, such as at least 98%, sequence identity. These terms also encompasspolypeptides derived from amino acids which are allelic variations ofthe PRL-1 or PRL-3 nucleic acid sequence.

Where reference is made to the “activity” or “biological activity” of apolypeptide such as PRL-1 and PRL-3, these terms are intended to referto the metabolic or physiological function of PRL-1 and PRL-3, includingsimilar activities or improved activities or these activities withdecreased undesirable side effects. Also included are antigenic andimmunogenic activities of PRL-1 and PRL-3. Examples of such activities,and methods of assaying and quantifying these activities, are known inthe art, and are described in detail elsewhere in this document.

Antibodies

The terms “antibody” and “immunoglobulin”, as used in this document, maybe employed interchangeably where the context permits. These terminclude fragments of proteolytically-cleaved or recombinantly-preparedportions of an antibody molecule that are capable of selectivelyreacting with or recognising PRL-1 or PRL-3 or an epitope thereof, suchas an epitope of PRL-1 bound by 269 or an epitope of PRL-3 bound by 223or 318.

Epitopes of PRL-1 include TYKNMR (SEQ ID NO: 13) and TLNKFI (SEQ ID NO:14). Epitopes of PRL-3 include KAKFYN (SEQ ID NO: 35) and HTHKTR (SEQ IDNO: 39).

Non limiting examples of such proteolytic and/or recombinant fragmentsinclude Fab, F (ab′) 2, Fab′, Fv fragments, and single chainantibodies(scFv) containing a VL and VH domain joined by a peptidelinker. These Fvs may be covalently or non-covalently linked to formantibodies having two or more binding sites.

By “ScFv molecules” we mean molecules wherein the VH and VL partnerdomains are linked via a flexible oligopeptide. A general review of thetechniques involved in the synthesis of antibody fragments which retaintheir specific binding sites is to be found in Winter & Milstein (1991)Nature 349, 293-299.

Whole antibodies, and F(ab′) 2 fragments are “bivalent”. By “bivalent”we mean that the said antibodies and F(ab′) fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent having only one antigen combining site.

The anti-PRL antibody may comprise a high affinity antibody with an offrate from 10⁻²s⁻¹ to 10⁻⁴s⁻¹. The off rate may be about 2×10⁻⁴s⁻¹.

The term “off-rate” as used in this document refers to the dissociationrate (k_(off)) of an antibody such as an anti-PRL antibody disclosedhere. It may be measured using BIAevaluation software (Pharmacia). A lowoff rate is desirable as it reflects the affinity of an Fab fragment foran antigen.

The term “affinity” is defined in terms of the dissociation rate oroff-rate (k_(off)) of a an antibody such as an anti-PRL antibody. Thelower the off-rate the higher the affinity that a an antibody such as ananti-PRL antibody has for an antigen such as PRL-1 or PRL-3.

The anti-PRL antibody may comprise a peptide per se or form part of afusion protein.

The anti-PRL antibodies described here include any antibody thatcomprise PRL-1 or PRL-3 binding activity, such as binding ability tointracellular PRL-1 or PRL-3 or binding to the same epitope bound by269, 223 or 318 as the case may be, including TYKNMR (SEQ ID NO: 13),TLNKFI (SEQ ID NO: 14), KAKFYN (SEQ ID NO: 35) and HTHKTR (SEQ ID NO:39).

The anti-PRL antibodies also include the entire or whole antibody,whether mouse, humanised or human, such antibody derivatives andbiologically-active fragments. These may include antibody fragments withPRL-1 or PRL-3 binding activity that have amino acid substitutions orhave sugars or other molecules attached to amino acid functional groups,etc.

The anti-PRL antibody may comprise isolated antibody or purifiedantibody. It may be obtainable from or produced by any suitable source,whether natural or not, or it may be a synthetic anti-PRL antibody, asemi-synthetic anti-PRL antibody, a derivatised anti-PRL antibody or arecombinant anti-PRL antibody.

Where the anti-PRL antibody is a non-native anti-PRL antibody, it mayinclude at least a portion of which has been prepared by recombinant DNAtechniques or an anti-PRL antibody produced by chemical synthesistechniques or combinations thereof.

The term “derivative” as used in this document includes chemicalmodification of an anti-PRL antibody. Illustrative of such modificationswould be replacement of hydrogen by an alkyl, acyl, or amino group, forexample. The sequence of the anti-PRL antibody may be the same as thatof the naturally occurring form or it may be a variant, homologue,fragment or derivative thereof.

Antibody Variable Regions

The term “variable region”, as used in this document, refers to thevariable regions, or domains, of the light chains (VL) and heavy chains(VH) which contain the determinants for binding recognition specificityand for the overall affinity of the antibody against PRL-1 or PRL-3 (orvariant, homologue, fragment or derivative), as the case may be.

The variable domains of each pair of light (VL) and heavy chains (VH)are involved in antigen recognition and form the antigen binding site.The domains of the light and heavy chains have the same generalstructure and each domain has four framework (FR) regions, whosesequences are relatively conserved, connected by three complementaritydetermining regions (CDRs). The FR regions maintain the structuralintegrity of the variable domain. The CDRs are the polypeptide segmentswithin the variable domain that mediate binding of the antigen.

The term “constant region”, as used in this document, refers to thedomains of the light (CL) and heavy (CH) chain of the antibody (orvariant, homologue, fragment or derivative) which provide structuralstability and other biological functions such as antibody chainassociation, secretion, transplacental mobility, and complement binding,but which are not involved with binding a PRL-1 or PRL-3 epitope. Theamino acid sequence and corresponding exon sequences in the genes of theconstant region will be dependent upon the species from which it isderived. However, variations in the amino acid sequence leading toallotypes are relatively limited for particular constant regions withina species. An “allotype” is an antigenic determinant (or epitope) thatdistinguishes allelic genes.

The variable region of each chain is joined to the constant region by alinking polypeptide sequence. The linkage sequence is coded by a “J”sequence in the light chain gene, and a combination of a “D” sequenceand a “J” sequence in the heavy chain gene.

Antibody 269, 223 and 318: Variable Region Sequences

Antibody 269

The nucleic acid sequence of the heavy chain of the variable region ofmonoclonal antibody 269 is as follows (SEQ ID NO: 1):

GGGAATTCATGAAATGCAGCTGGGTTATTCTCTTCCTGTTTTCAGTAACTGCAGGTGTCCACTCCCAGGTCCAGTTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACTTTTACTAGTTATCGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCACTGGTTATACTGAGTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTTCAAGCTATGGTAACTTCGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGAGAGTCAGTCCTTCCCAAATGTCTTCCCCC TCGTAAGCTTGGGA

The amino acid sequence of the heavy chain of the variable region ofmonoclonal antibody 269 is as follows (SEQ ID NO: 2):

EFMKCSWVILFLFSVTAGVHSQVQFQQSGAELAKPGASVKMSCKASGYTFTSYRMHWVKQRPGQGLEWIGYINPSTGYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCSSYGNFGYWGQGTTLTVSSESQSFPNVFPL VSLG

The nucleic acid sequence of the light chain of the variable region ofmonoclonal antibody 269 is as follows (SEQ ID NO: 3):

CTGTCTACTGCTCTCTGGTGAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGACATTGGTAGTAGCTTAAACTGGCTTCAGCAGAAAGCAGATGGAACCATTAAACGCCTGATCTATGCCACATCCAGTTTAGATTCTGGTGTCCCCAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGTAGACTATTACTGTCTACAATATGCTAGTTCTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCTCACTGGAGATCCTGCAGATCACGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT

The amino acid sequence of the light chain of the variable region ofmonoclonal antibody 269 is as follows (SEQ ID NO: 4):

VYCSLVRVSLTCRASQDIGSSLNWLQQKADGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPWTFGGGTKLEIKRADAAPHWRSCRSRELWLHHLSSSSRHLMSS

Antibody 223

The nucleic acid sequence of the heavy chain of the variable region ofmonoclonal antibody 223 is as follows (SEQ ID NO: 5):

GGGAATTCATGGAATGGAGCTGGGTTATTCTCTTCCTCCTGTCAATAATTGCAGGTGTCCATTGCCAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATGTTAATACTGAGTACAATGAGAAGTTCAGGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGTGAGGAGAGGAATTACCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTCTATCCCTTGGTCCCTGGAAGCTTGGGA

The amino acid sequence of the heavy chain of the variable region ofmonoclonal antibody 223 is as follows (SEQ ID NO: 6):

EFMEWSWVILFLLSIIAGVHCQVQLQQSGPELVKPGASVRISCKASGYTFTSYYIHWVKQRPGQGLEWIGWIYPGNVNTEYNEKFRGKATLTADKSSSTAYMQLSSLTSEDSAVYFCASEERNYPWFAYWGQGTLVTVSAAKTTPPP VYPLVPGSLG

The nucleic acid sequence of the light chain of the variable region ofmonoclonal antibody 223 is as follows (SEQ ID NO: 7):

TGGGAATTCATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGCTCCACTGGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGAAGATGATGGTGAAAATTATATGAACTGGTACCAACAGAAACCAGGACAGTCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCA TCCAGTAAGCTTGGG

The amino acid sequence of the light chain of the variable region ofmonoclonal antibody 223 is as follows (SEQ ID NO: 8):

WEFMETDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVEDDGENYMNWYQQKPGQSPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIKRADAAPTVSIFPPSSK LG

Antibody 318

The nucleic acid sequence of the heavy chain of the variable region ofmonoclonal antibody 318 is as follows (SEQ ID NO: 9):

GGGAATTCATGGAATGGAGCTGGGTTTTCCTCTTCCTCCTGTCAATAATTGCAGGTGTCCATTGCCAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAACTACTATATGCACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATGTTAATACTTATTACAATGAGAAGTTCAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGTGAGGAGAGAATTACCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTATCCCCTGGTCCCTGGAAGCTTGGGA

The amino acid sequence of the heavy chain of the variable region ofmonoclonal antibody 318 is as follows (SEQ ID NO: 10):

EFMEWSWVFLFLLSIIAGVHCQVQLQQSGPELVKPGASVRISCKASGYTFTNYYMHWVKQRPGQGLEWIGWIYPGNVNTYYNEKFRARPH.LQTNPPAQPTCSSAA.PLRTLRSISVQVRRELPLVCLLGPRDSGHCLCSQNDTPIR LSPGPWKLG

The nucleic acid sequence of the light chain of the variable region ofmonoclonal antibody 318 is as follows (SEQ ID NO: 11):

ACTAGTCGACATGGAGTCAGACACACTGCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCC ATAAGCTTGGGA

The amino acid sequence of the light chain of the variable region ofmonoclonal antibody 318 is as follows (SEQ ID NO: 12):

LVDMESDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK

Anti-PRL1 and anti-PRL3 antibodies, according to the methods andcompositions described here, may be generated from these variable regionsequences by methods known in the art. For example, the heavy and lightchain sequences may be recombined into a constant sequence for a chosenantibody, through recombinant genetic engineering techniques which areknown to the skilled person.

Constant region sequences are known in the art, and are available from anumber of databases, such as the IMGT/LIGM-DB database (described inGiudicelli et al, 2006, Nucleic Acids Research 34(DatabaseIssue):D781-D784 and LeFranc et al (1995) LIGM-DB/IMGT: An IntegratedDatabase of Ig and TcR, Part of the Immunogenetics Database. Annals ofthe New York Academy of Sciences 764 (1), 47-47doi:10.1111/j.1749-6632.1995.tb55805.x) and the IMGT/GENE-DB database(described in Giudicelli et al, 2005, Nucleic Acids Res. 2005 Jan. 1;33(Database issue):D256-61). IMGT/LIGM-DB and IMGT/GENE-DB are part ofthe ImMunoGeneTics Database located at www.ebi.ac.uk/imgt/.

Methods for combining variable regions with given sequences and constantregions to produce whole antibodies are known in the art and aredescribed for example in Example 16 and in Hanson et al., (2006).Respiratory Research, 7:126. Fragments of whole antibodies such as Fv,F(ab′) and F(ab′)₂ fragments or single chain antibodies (scFv) may beproduced by means known in the art.

Using the disclosed sequences and the methods described in theliterature, for example, the heavy and light chains of the variableregion of antibody 318, having the sequences shown above, may betransgenically fused to a mouse IgG constant region sequence to producea mouse monoclonal anti-PRL-3 antibody. Similarly, the 318 variableregion may be recombinantly expressed with the constant region of ahuman IgG antibody to produce a humanized anti-PRL-3 antibody. Variableregions of 223 and 269 antibodies may be engineered with mouse or humanIgG constant regions to produce mouse monoclonal or humanized antibodiescapable of binding to PRL-1 polypeptide.

Detection and Diagnostic Methods

Detection of Expression of PRL-1 and PRL-3

Expression of PRL-1 and PRL-3 in cancer tissue is up-regulated whencompared to normal tissue.

Accordingly, we provide for a method of diagnosis of cancer, includingmetastatic, aggressive or invasive cancer, comprising detectingmodulation of expression of PRL-1 and PRL-3, such as up-regulation ofexpression of PRL-1 and PRL-3 in a cell or tissue of an individual.

The method may comprise use of the anti-PRL antibodies described in thisdocument. The anti-PRL antibodies may be used in immunoassays to detectand assay the quantity of PRL-1 or PRL-3 in a biological sample, andhence provide an indication of the level of expression of PRL-1 or PRL-3in a cell, tissue, organ or individual from which the sample is derived.Immunoassays include ELISA, Western Blot, etc, and methods of employingthese to assess PRL-1 and PRL-3 expression are known to the skilledreader.

Detection of PRL-1 and PRL-3 expression, activity or amount may be usedto provide a method of determining the proliferative state of a cell.Thus, a proliferative cell is one with high levels of PRL-1 and PRL-3expression, activity or amount compared to a normal cell. Similarly, anon-proliferative cell may be one with low levels PRL-1 and PRL-3expression, activity or amount compared to a normal cell.

Such detection may also be used to determine whether a cell will becomeinvasive or aggressive. Thus, detection of a high level of PRL-1 andPRL-3 expression, amount or activity of PRL-1 and PRL-3 in the cell mayindicate that the cell is likely to be or become aggressive, metastaticor invasive. Similarly, if a cell has a low level of PRL-1 and PRL-3expression, amount or activity, the cell is not or is not likely to beaggressive, metastatic or invasive.

It will be appreciated that as the level of PRL-1 and PRL-3 varies withthe aggressiveness of a tumour, that detection of PRL-1 and PRL-3expression, amount or activity may also be used to predict a survivalrate of an individual with cancer, i.e., high levels of PRL-1 and PRL-3indicating a lower survival rate or probability and low levels of PRL-1and PRL-3 indicating a higher survival rate or probability, both ascompared to individuals or cognate populations with normal levels ofPRL-1 and PRL-3. Detection of expression, amount or activity of PRL-1and PRL-3 may therefore be used as a method of prognosis of anindividual with cancer.

Detection of PRL-1 and PRL-3 expression, amount or level may be used todetermine the likelihood of success of a particular therapy in anindividual with a cancer. It may be used in a method of determiningwhether a tumour in an individual is, or is likely to be, an invasive ormetastatic tumour.

The diagnostic methods described in this document may be combined withthe therapeutic methods described. Thus, we provide for a method oftreatment, prophylaxis or alleviation of cancer in an individual, themethod comprising detecting modulation of expression, amount or activityof PRL-1 and PRL-3 in a cell of the individual and administering anappropriate therapy to the individual based on the aggressiveness of thetumour.

Typically, physical examination X-rays are used for the detection ofcancer. A biopsy of the tumour is typically taken for histopathologicalexamination for the diagnosis of cancer. Detection of PRL-1 and PRL-3expression, amount or activity can be used to diagnose, or furtherconfirm the diagnosis of, cancer, along with the standardhistopathological procedures. This may be especially useful when thehistopathological analysis does not yield a clear result.

The presence and quantity of PRL-1 and PRL-3 polypeptides and nucleicacids may be detected in a sample as described in further detail below.Thus, the PRL-1 and PRL-3 associated diseases, including cancer, can bediagnosed by methods comprising determining from a sample derived from asubject an abnormally decreased or increased expression, amount oractivity, such as a increased expression, amount or activity, of thePRL-1 and PRL-3 polypeptide or PRL-1 and PRL-3 mRNA.

The sample may comprise a cell or tissue sample from an organism orindividual suffering or suspected to be suffering from a diseaseassociated with increased, reduced or otherwise abnormal PRL-1 and PRL-3expression, amount or activity, including spatial or temporal changes inlevel or pattern of expression, amount or activity. The level or patternof expression, amount or activity of PRL-1 and PRL-3 in an organismsuffering from or suspected to be suffering from such a disease may beusefully compared with the level or pattern of expression, amount oractivity in a normal organism as a means of diagnosis of disease.

The sample may comprise a cell or tissue sample from an individualsuffering or suspected to be suffering from cancer, such as a relevanttissue or cell sample.

In some embodiments, an increased level of expression, amount oractivity of PRL-1 and PRL-3 is detected in the sample. The level ofPRL-1 and PRL-3 may be increased to a significant extent when comparedto normal cells, or cells known not to be cancerous. Such cells may beobtained from the individual being tested, or another individual, suchas those matched to the tested individual by age, weight, lifestyle,etc.

In some embodiments, the level of expression, amount or activity ofPRL-1 and PRL-3 is increased by 10%, 20%, 30% or 40% or more. In someembodiments, the level of expression, amount or activity of PRL-1 andPRL-3 is increased by 45% or more, such as 50% or more, as judged bycDNA hybridisation.

The expression, amount or activity of PRL-1 and PRL-3 may be detected ina number of ways, as known in the art, and as described in furtherdetail below. Typically, the amount of PRL-1 and PRL-3 in a sample oftissue from an individual is measured, and compared with a sample froman unaffected individual. Both PRL-1 and PRL-3 nucleic acid, as well asPRL-1 and PRL-3 polypeptide levels may be measured.

Detection of the amount, activity or expression of PRL-1 and PRL-3 maybe used to grade the cancer. For example, a high level of amount,activity or expression of PRL-1 and PRL-3 may indicate an aggressive,invasive or metastatic cancer. Similarly, a low level of amount,activity or expression of PRL-1 and PRL-3 may indicate a non-aggressive,non-invasive or non-metastatic cancer. Such a grading system may be usedin conjunction with established grading systems.

Levels of PRL-1 and PRL-3 gene expression may be determined using anumber of different techniques.

Measuring Expression of PRL-1 and PRL-3 at the RNA Level

PRL-1 and PRL-3 gene expression can be detected at the RNA level.

In one embodiment therefore, we disclose a method of detecting thepresence of a nucleic acid comprising a PRL-1 and PRL-3 nucleic acid ina sample, by contacting the sample with at least one nucleic acid probewhich is specific for the PRL-1 and PRL-3 nucleic acid and monitoringsaid sample for the presence of the PRL-1 and PRL-3 nucleic acid. Forexample, the nucleic acid probe may specifically bind to the PRL-1 andPRL-3 nucleic acid, or a portion of it, and binding between the twodetected; the presence of the complex itself may also be detected.

RNA detection of expression of PRL-1 and PRL-3 may be used to supplementpolypeptide expression assays, as described below, which may employ theanti-PRL antibodies described here.

Thus, in one embodiment, the amount of PRL-1 and PRL-3 nucleic acid inthe form of PRL-1 and PRL-3 mRNA may be measured in a sample. PRL-1 andPRL-3 mRNA may be assayed by in situ hybridization, Northern blottingand reverse transcriptase—polymerase chain reaction. Nucleic acidsequences may be identified by in situ hybridization, Southern blotting,single strand conformational polymorphism, PCR amplification andDNA-chip analysis using specific primers. (Kawasaki, 1990; Sambrook,1992; Lichter et al, 1990; Orita et al, 1989; Fodor et al., 1993; Peaseet al., 1994).

PRL-1 and PRL-3 RNA may be extracted from cells using RNA extractiontechniques including, for example, using acid phenol/guanidineisothiocyanate extraction (RNAzol B; Biogenesis), or RNeasy RNApreparation kits (Qiagen). Typical assay formats utilising ribonucleicacid hybridisation include nuclear run-on assays, RT-PCR and RNaseprotection assays (Melton et al., Nuc. Acids Res. 12:7035. Methods fordetection which can be employed include radioactive labels, enzymelabels, chemiluminescent labels, fluorescent labels and other suitablelabels.

Each of these methods allows quantitative determinations to be made, andare well known in the art. Decreased or increased PRL-1 and PRL-3expression, amount or activity can therefore be measured at the RNAlevel using any of the methods well known in the art for thequantitation of polynucleotides. Any suitable probe from a PRL-1 andPRL-3 sequence, for example, any portion of a suitable human PRL-1 andPRL-3 sequence may be used as a probe. Sequences for designing PRL-1 andPRL-3 probes may include a sequence having accession numberNM_(—)015472, or a portion thereof.

Typically, RT-PCR is used to amplify RNA targets. In this process, thereverse transcriptase enzyme is used to convert RNA to complementary DNA(cDNA) which can then be amplified to facilitate detection.

Many DNA amplification methods are known, most of which rely on anenzymatic chain reaction (such as a polymerase chain reaction, a ligasechain reaction, or a self-sustained sequence replication) or from thereplication of all or part of the vector into which it has been cloned.

Many target and signal amplification methods have been described in theliterature, for example, general reviews of these methods in Landegren,U. et al., Science 242:229-237 (1988) and Lewis, R., Genetic EngineeringNews 10:1, 54-55 (1990).

For example, the polymerase chain reaction may be employed to detectPRL-1 and PRL-3 mRNA.

The “polymerase chain reaction” or “PCR” is a nucleic acid amplificationmethod described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202.PCR can be used to amplify any known nucleic acid in a diagnosticcontext (Mok et al., 1994, Gynaecologic Oncology 52:247-252).Self-sustained sequence replication (3SR) is a variation of TAS, whichinvolves the isothermal amplification of a nucleic acid template viasequential rounds of reverse transcriptase (RT), polymerase and nucleaseactivities that are mediated by an enzyme cocktail and appropriateoligonucleotide primers (Guatelli et al., 1990, Proc. Natl. Acad. Sci.USA 87:1874). Ligation amplification reaction or ligation amplificationsystem uses DNA ligase and four oligonucleotides, two per target strand.This technique is described by Wu, D. Y. and Wallace, R. B., 1989,Genomics 4:560. In the Q13 Replicase technique, RNA replicase for thebacteriophage Q13, which replicates single-stranded RNA, is used toamplify the target DNA, as described by Lizardi et al., 1988,Bio/Technology 6:1197.

A PCR procedure basically involves: (1) treating extracted DNA to formsingle-stranded complementary strands; (2) adding a pair ofoligonucleotide primers, wherein one primer of the pair is substantiallycomplementary to part of the sequence in the sense strand and the otherprimer of each pair is substantially complementary to a different partof the same sequence in the complementary antisense strand; (3)annealing the paired primers to the complementary sequence; (4)simultaneously extending the annealed primers from a 3′ terminus of eachprimer to synthesize an extension product complementary to the strandsannealed to each primer wherein said extension products after separationfrom the complement serve as templates for the synthesis of an extensionproduct for the other primer of each pair; (5) separating said extensionproducts from said templates to produce single-stranded molecules; and(6) amplifying said single-stranded molecules by repeating at least oncesaid annealing, extending and separating steps.

Reverse transcription-polymerase chain reaction (RT-PCR) may beemployed. Quantitative RT-PCR may also be used. Such PCR techniques arewell known in the art, and may employ any suitable primer from a PRL-1and PRL-3 sequence.

Alternative amplification technology can also be exploited. For example,rolling circle amplification (Lizardi et al., 1998, Nat Genet 19:225) isan amplification technology available commercially (RCAT™) which isdriven by DNA polymerase and can replicate circular oligonucleotideprobes with either linear or geometric kinetics under isothermalconditions. A further technique, strand displacement amplification (SDA;Walker et al., 1992, Proc. Natl. Acad. Sci. USA 80:392) begins with aspecifically defined sequence unique to a specific target.

Measuring Expression of PRL-1 and PRL-3 at the Polypeptide Level

PRL-1 and PRL-3 expression can be detected at the polypeptide level.

In a further embodiment, therefore, PRL-1 and PRL-3 expression, amountor activity may be detected by detecting the presence or amount of PRL-1and PRL-3 polypeptide in a sample. This may be achieved by usingmolecules which bind to PRL-1 and PRL-3 polypeptide. Suitablemolecules/agents which bind either directly or indirectly to the PRL-1and PRL-3 polypeptide in order to detect its presence include naturallyoccurring molecules such as peptides and proteins, for exampleantibodies, or they may be synthetic molecules.

Thus, we disclose a method of detecting the presence of a PRL-1 andPRL-3 polypeptide by contacting a cell sample with an antibody capableof binding the polypeptide and monitoring said sample for the presenceof the polypeptide.

For example, the PRL-1 and PRL-3 polypeptide may be detected using ananti-PRL-1 and PRL-3 antibody as described here. Such antibodies may bemade by means described in detail in this document. In a specificexample, an anti-PRL-1 antibody may comprise an antibody capable ofbinding to the same epitope as monoclonal antibody 269. This may includemonoclonal antibody 269 itself, an antibody comprising a variable regionof antibody 269, or a humanised monoclonal antibody 269.

Similarly, an anti-PRL-3 antibody may comprise an antibody capable ofbinding to the same epitope as monoclonal antibody 223 or 318. This mayinclude monoclonal antibody 223 or 318 itself, an antibody comprising avariable region of antibody 223 or 318, or a humanised monoclonalantibody 223 or 318.

The assay may conveniently be achieved by monitoring the presence of acomplex formed between the antibody and the polypeptide, or monitoringthe binding between the polypeptide and the antibody. Methods ofdetecting binding between two entities are known in the art, and includeFRET (fluorescence resonance energy transfer), surface plasmonresonance, etc.

Standard laboratory techniques such as immunoblotting as described abovecan be used to detect altered levels of PRL-1 and PRL-3 protein, ascompared with untreated cells in the same cell population.

Gene expression may also be determined by detecting changes inpost-translational processing of PRL-1 and PRL-3 polypeptides orpost-transcriptional modification of PRL-1 and PRL-3 nucleic acids. Forexample, differential phosphorylation of PRL-1 and PRL-3 polypeptides,the cleavage of PRL-1 and PRL-3 polypeptides or alternative splicing ofPRL-1 and PRL-3 RNA, and the like may be measured. Levels of expressionof gene products such as PRL-1 and PRL-3 polypeptides, as well as theirpost-translational modification, may be detected using proprietaryprotein assays or techniques such as 2D polyacrylamide gelelectrophoresis.

Assay techniques that can be used to determine levels of PRL-1 and PRL-3protein in a sample derived from a host are well-known to those of skillin the art. Antibodies can be assayed for immunospecific binding by anymethod known in the art.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA, sandwich immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays and protein A immunoassays. Such assays are routine in theart (see, for example, Ausubel et al., eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which isincorporated by reference herein in its entirety).

The specimen may be assayed for polypeptides/proteins byimmunohistochemical and immunocytochemical staining (see generallyStites and Terr, Basic and Clinical Immunology, Appleton and Lange,1994), ELISA, RIA, immunoblots, Western blotting, immunoprecipitation,functional assays and protein truncation test. Other assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

ELISA assays are well known to those skilled in the art. Both polyclonaland monoclonal antibodies may be used in the assays. Where appropriateother immunoassays, such as radioimmunoassays (RIA) may be used as areknown to those in the art. Available immunoassays are extensivelydescribed in the patent and scientific literature. See, for example,U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 as well asSambrook et al, 1992.

Diagnostic Kits

We also provide diagnostic kits for detecting cancer in an individual,or susceptibility to cancer in an individual.

The diagnostic kit may comprise means for detecting expression, amountor activity of PRL-1 or PRL-3 in the individual, by any means asdescribed in this document. The diagnostic kit may therefore compriseany one or more of the following: an anti-PRL antibody, an antibodycapable of binding to the same epitope as monoclonal antibody 269, 223or 318, monoclonal antibody 269, an antibody comprising a variableregion of antibody 269, or a humanised monoclonal antibody 269;monoclonal antibody 223, an antibody comprising a variable region ofantibody 223, or a humanised monoclonal antibody 223; monoclonalantibody 318, an antibody comprising a variable region of antibody 318,or a humanised monoclonal antibody 318.

The diagnostic kit may comprise instructions for use, or other indicia.The diagnostic kit may further comprise means for treatment orprophylaxis of breast cancer, such as any of the compositions describedin this document, or any means known in the art for treating breastcancer. In particular, the diagnostic kit may comprise an anti-PRL1 oranti-PRL3 antibody as described, for example obtained by screening.

Prophylactic and Therapeutic Methods

We disclose methods of treating an abnormal conditions, such as cancer,related to excessive amounts of PRL-1 and PRL-3 expression or activity.Methods of preventing cancer (i.e., prophylaxis) also suitably employthe same or similar approaches.

In general terms, our methods involve manipulation of cancer cells, bymodulating (such as down-regulating) the expression, amount or activityof PRL-1 and PRL-3 in the cell. The methods may involve destroying oreradicating cancer cells. The cancer cells may comprise PRL-1 and/orPRL-3 expressing cancer cells. The cancer cells may be ones whichover-express PRL-1 and/or PRL-3, compared to non-cancerous cells. Ourmethods may comprise exposing a patient to an anti-PRL antibody, such asan anti-PRL-1 antibody or an anti-PRL-3 antibody, or both. Theanti-PRL-1 antibody may comprise a humanised anti-PRL-1 antibody;likewise, the anti-PRL-3 antibody may comprise a humanised anti-PRL-3antibody.

The cancer cells may be from PRL-1 and/or PRL-3 positive cancerpatients. Thus, our methods may comprise eradicating PRL-1 and/orPRL-3-over-expressing cancer cells from PRL-3/-1-positive cancerpatients.

Our methods may therefore comprise eradicating PRL-1 and/or PRL-3over-expressing cells from PRL-3/-1-positive cancer patients usingPRL-3/-1 humanised antibodies.

A step of detecting modulated PRL-1 and PRL-3 expression, amount oractivity in a cell may be conducted before or after the manipulationstep. The detection step may detect up-regulated or down-regulated PRL-1and PRL-3 expression, amount or activity. Any of the methods ofmodulating or down-regulating PRL-1 and PRL-3, as described in detailelsewhere in this document, may be used.

In particular, the method may comprise exposing the cell to ananti-PRL-1 or anti-PRL-3 antibody capable of specifically binding toPRL-1 or PRL-3. Anti-PRL antibodies and methods of administering themare described in detail elsewhere in this document.

According to our methods, the cancer cell becomes non-cancerous or theinvasive or metastatic cancer cell becomes non-invasive ornon-metastatic as a result of the manipulation. The cancer may inparticular comprise a cancer such as an invasive or metastatic cancerselected from the group consisting of: colorectal cancer, ovariancancer, breast cancer, liver cancer, pancreatic cancer, prostate cancer,gastric cancer, lung cancer, penis cancer, cervical cancer, braincancer, esophageal cancer, bladder carcinoma, kidney renal cellcarcinoma, ovary lymphoma and skin melanoma.

As PRL-1 and PRL-3 is associated with aggressiveness and invasiveness ofcancer, the level of PRL-1 and PRL-3 may be detected in a cell of anindividual with cancer, in a cancer or non-cancer cell, and theaggressiveness of the cancer assessed. A high level of PRL-1 and PRL-3amount, expression or activity compared with a normal cell indicates anaggressive or invasive cancer, and a stronger or harsher therapy maytherefore be required and chosen. Similarly, a lower level may indicatea less aggressive or invasive therapy.

The approaches described here may be used for therapy of any PRL-1 andPRL-3 related disease in general. PRL-1 and PRL-3 related diseasesinclude proliferative diseases and in particular include cancer. Forexample, a PRL-1 and PRL-3 related disease may include metastaticcancer, invasive cancer or aggressive cancer.

The methods and compositions described here suitably enable animprovement in a measurable criterion in an individual to whom thetreatment is applied, compared to one who has not received thetreatment.

For this purpose, a number of criteria may be designated, which reflectthe progress of cancer or the well-being of the patient. Useful criteriamay include tumour size, tumour dimension, largest dimension of tumour,tumour number, presence of tumour markers (such as alpha-feto protein),degree or number of metastates, etc.

Thus, as an example, a treated individual may show a decrease in tumoursize or number as measured by an appropriate assay or test. A treatedindividual may for example show a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or moredecrease in tumour size of a particular tumour, or decrease in tumournumber, or both, compared to an individual who has not been treated.

For example, a PRL-1 and PRL-3 related disease may be defined as being“treated” if a condition associated with the disease is significantlyinhibited (i.e., by 50% or more) relative to controls. The inhibitionmay be by at least 75% relative to controls, such as by 90%, by 95% or100% relative to controls. The condition may comprise cellproliferation, or it may comprise cell cycle time, cell number, cellmigration, cell invasiveness, tumour formation, tumour metastasis,tumour spread, etc. By the term “treatment” we mean to also includeprophylaxis or alleviation of cancer.

The term proliferative disorder is used herein in a broad sense toinclude any disorder that requires control of the cell cycle. Inparticular, a proliferative disorder includes malignant andpre-neoplastic disorders. The methods and compositions described hereare especially useful in relation to treatment or diagnosis ofadenocarcinomas such as: small cell lung cancer, and cancer of thekidney, uterus, prostrate, bladder, ovary, colon and breast. Forexample, malignancies which may be treatable include acute and chronicleukemias, lymphomas, myelomas, sarcomas such as Fibrosarcoma,myxosarcoma, liposarcoma, lymphangioendotheliosarcoma, angiosarcoma,endotheliosarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,lymphangiosarcoma, synovioma, mesothelioma, leimyosarcoma,rhabdomyosarcoma, colon carcinoma, ovarian cancer, prostate cancer,pancreatic cancer, breast cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,choriocarcinoma, renal cell carcinoma, hepatoma, bile duct carcinomaseminoma, embryonal carcinoma, cervical cancer, testicular tumour, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, ependymoma, pinealoma, hemangioblastoma,acoustic neuoma, medulloblastoma, craniopharyngioma, oligodendroglioma,menangioma, melanoma, neutroblastoma and retinoblastoma.

The antibody approach to therapy involving use of anti-PRL antibodiesmay be combined with other approaches for therapy of such disordersincluding expression of anti-sense constructs directed against PRL-1 andPRL-3 polynucleotides as described here, and administering them totumour cells, to inhibit gene function and prevent the tumour cell fromgrowing or progressing.

Anti-sense constructs may be used to inhibit gene function to preventgrowth or progression in a proliferative cell. Antisense constructs,i.e., nucleic acid, such as RNA, constructs complementary to the sensenucleic acid or mRNA, are described in detail in U.S. Pat. No. 6,100,090(Monia et al.), and Neckers et al., 1992, Crit Rev Oncog 3(1-2):175-231,the teachings of which document are specifically incorporated byreference.

In a particular example, cancer may be treated or prevented by reducingthe amount, expression or activity of PRL-1 and PRL-3 in whole or inpart, for example by siRNAs capable of binding to and destroying PRL-1and PRL-3 mRNA.

RNA interference (RNAi) is a method of post transcriptional genesilencing (PTGS) induced by the direct introduction of double-strandedRNA (dsRNA) and has emerged as a useful tool to knock out expression ofspecific genes in a variety of organisms. RNAi is described by Fire etal., Nature 391:806-811 (1998). Other methods of PTGS are known andinclude, for example, introduction of a transgene or virus. Generally,in PTGS, the transcript of the silenced gene is synthesised but does notaccumulate because it is rapidly degraded. Methods for PTGS, includingRNAi are described, for example, in the Ambion.com world wide web site,in the directory “/hottopics/”, in the “rnai” file.

Suitable methods for RNAi in vitro are described herein. One such methodinvolves the introduction of siRNA (small interfering RNA). Currentmodels indicate that these 21-23 nucleotide dsRNAs can induce PTGS.Methods for designing effective siRNAs are described, for example, inthe Ambion web site described above. RNA precursors such as ShortHairpin RNAs (shRNAs) can also be encoded by all or a part of the PRL-1and PRL-3 nucleic acid sequence.

Alternatively, double-stranded (ds) RNA is a powerful way of interferingwith gene expression in a range of organisms that has recently beenshown to be successful in mammals (Wianny and Zernicka-Goetz, 2000, NatCell Biol 2:70-75). Double stranded RNA corresponding to the sequence ofa PRL-1 and PRL-3 polynucleotide can be introduced into or expressed inoocytes and cells of a candidate organism to interfere with PRL-1 andPRL-3 activity.

Other methods of modulating PRL-1 and PRL-3 gene expression are known tothose skilled in the art and include dominant negative approaches.Again, these may be combined with antibody therapy using anti-PRLantibodies. Thus, another approach is to use non-functional variants ofPRL-1 and PRL-3 polypeptide in this document that compete with theendogenous gene product resulting in inhibition of function.

PRL-1 and PRL-3 gene expression may also be modulated by as introducingpeptides or small molecules which inhibit gene expression or functionalactivity. Such peptides or small molecules may be administered incombination with anti-PRL antibodies for the treatment of cancer such asmetastatic cancer.

Thus, compounds identified by assays as binding to or modulating, suchas down-regulating, the amount, activity or expression of PRL-1 andPRL-3 polypeptide may be administered to tumour or proliferative cellsto prevent the function of PRL-1 and PRL-3 polypeptide. Such a compoundmay be administered along with a pharmaceutically acceptable carrier inan amount effective to down-regulate expression or activity PRL-1 andPRL-3, or by activating or down-regulating a second signal whichcontrols PRL-1 and PRL-3 expression, activity or amount, and therebyalleviating the abnormal condition.

Alternatively, gene therapy may be employed to control the endogenousproduction of PRL-1 and PRL-3 by the relevant cells such as cancer cellsin the subject. For example, a polynucleotide encoding a PRL-1 and PRL-3siRNA or a portion of this may be engineered for expression in areplication defective retroviral vector, as discussed below. Theretroviral expression construct may then be isolated and introduced intoa packaging cell transduced with a retroviral plasmid vector containingRNA encoding an anti-PRL-1 and PRL-3 siRNA such that the packaging cellnow produces infectious viral particles containing the sequence ofinterest. These producer cells may be administered to a subject forengineering cells in vivo and regulating expression of the PRL-1 andPRL-3 polypeptide in vivo. For overview of gene therapy, see Chapter 20,Gene Therapy and other Molecular Genetic-based Therapeutic Approaches,(and references cited therein) in Human Molecular Genetics, T Strachanand A P Read, BIOS Scientific Publishers Ltd (1996).

In some embodiments, the level of PRL-1 and PRL-3 is decreased in acancer cell. Furthermore, in such embodiments, treatment may be targetedto, or specific to, such cancer cells. The expression of PRL-1 and PRL-3may be specifically decreased only in diseased cells (i.e., those cellswhich are cancerous), and not substantially in other non-diseased cells.In these methods, expression of PRL-1 and PRL-3 may be not substantiallyreduced in other cells, i.e., cells which are not cancer cells. Thus, insuch embodiments, the level of PRL-1 and PRL-3 remains substantially thesame or similar in non-cancer cells in the course of or followingtreatment.

Polypeptide Sequences

It will be understood that polypeptide sequences disclosed here are notlimited to the particular sequences set forth in this document, but alsoinclude homologous sequences obtained from any source, for examplerelated cellular homologues, homologues from other species and variantsor derivatives thereof, provided that they have at least one of thebiological activities of an anti-PRL antibody, as the case may be.

This disclosure therefore encompasses variants, homologues orderivatives of the amino acid sequences set forth in this document, aswell as variants, homologues or derivatives of the amino acid sequencesencoded by the nucleotide sequences disclosed here. Such sequences aregenerally referred to as a “anti-PRL antibody” sequence.

Biological Activities

In some embodiments, the sequences comprise at least one biologicalactivity of an anti-PRL antibody, as the case may be.

The biological activity may comprise an immunological activity. Theanti-PRL antibody may comprise an identical or similar immunologicalactivity as compared to antibody 269, 223 ort 318, or their humanisedversions. By “immunological activity” we mean the capability of theanti-PRL antibody, to induce a specific immune response in appropriateanimals or cells on binding with a PRL-1 or PRL-3 antigen.

The biological activity may comprise antigen binding activity. Theanti-PRL antibody may bind to PRL-1 or an epitope thereof. The anti-PRLantibody may bind to the same epitope bound by antibody 269. Theanti-PRL antibody may bind to PRL-3 or an epitope thereof. The anti-PRLantibody may bind to the same epitope bound by antibody 223 or the sameepitope bound by antibody 318.

The anti-PRL antibody may bind to the antigen or epitope with the same,a reduced or elevated affinity or avidity. For example, the anti-PRLantibody may bind to the antigen or epitope with at least 10%, such as20%, such as 30%, 40% 50%, 60%, 70%, 80%, 90% or more, affinity oravidity compared to the cognate antibody, e.g., 269, 223 or 318 or theirhumanised counterparts, as the case may be.

The activity may include inhibition of cancer activity as for examplemeasured by reduction of tumour size or tumour number, or inhibition ofmetastatic activity, such as for example measured by the assaysdescribed in the Examples. The reduction or inhibition may beconveniently assayed by causing carcinogenesis in a test animal,administering the anti-PRL antibody to the animal and determining aneffect of the anti-PRL antibody as compared to a similar control animalthat has not been so treated. The Examples describe such an assay indetail.

The anti-PRL antibody may have tumour inhibition or metastasisinhibition activity that is the same as, reduced from, or elevated from,the cognate antibody. For example, the anti-PRL antibody may be at least10%, such as 20%, such as 30%, 40% 50%, 60%, 70%, 80%, 90% or more,effective compared to the cognate antibody, e.g., 269, 223 or 318 ortheir humanised counterparts, as the case may be. By this we mean that,say, if the cognate antibody is capable of reducing tumour number by 90%(see the Examples), the anti-PRL antibody may be capable of reducingtumour number by 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, etc, as compared to an untreated animal.

Other assays that detect antibody events can also be used, instead of,or in addition to, the assays described.

Homologues

The anti-PRL antibody polypeptides disclosed include homologoussequences obtained from any source, for example related viral/bacterialproteins, cellular homologues and synthetic peptides, as well asvariants or derivatives thereof. Thus polypeptides also include thoseencoding homologues of anti-PRL antibody from other species includinganimals such as mammals (e.g. mice, rats or rabbits), in particularhumans.

In the context of the present document, a homologous sequence orhomologue is taken to include an amino acid sequence which is at least60, 70, 80 or 90% identical, such as at least 95 or 98% identical at theamino acid level over at least 30, such as 50, 70, 90 or 100 amino acidswith a relevant polypeptide sequence, for example as shown in thesequence listing herein. In the context of this document, a homologoussequence is taken to include an amino acid sequence which is at least15, 20, 25, 30, 40, 50, 60, 70, 80 or 90% identical, such as at least 95or 98% identical at the amino acid level, such as over at least 15, 25,35, 50 or 100, such as 200, 300, 400 or 500 amino acids with thesequence of a relevant polypeptide. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentdocument homology may be expressed in terms of sequence identity. Thesequence identity may be determined relative to the entirety of thelength the relevant sequence, i.e., over the entire length or fulllength sequence of the relevant gene, for example.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,the default values may be used when using such software for sequencecomparisons. For example when using the GCG Wisconsin Bestfit package(see below) the default gap penalty for amino acid sequences is −12 fora gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). The GCG Bestfit program may be used.

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). The public default values for theGCG package, or in the case of other software, the default matrix, suchas BLOSUM62, may be used.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, such as % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Variants and Derivatives

The terms “variant” or “derivative” in relation to the amino acidsequences as described here includes any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) amino acids from or to the sequence. The resultant amino acidsequence may retain substantially the same activity as the unmodifiedsequence, such as having at least the same activity as the anti-PRLantibody polypeptides shown in this document, for example in thesequence listings. Thus, the key feature of the sequences—namely abilityto bind to PRL polypeptides or tumour reduction activity, as describedelsewhere—may be retained.

Polypeptides having the amino acid sequence shown in the Examples, orfragments or homologues thereof may be modified for use in the methodsand compositions described here. Typically, modifications are made thatmaintain the biological activity of the sequence. Amino acidsubstitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30substitutions provided that the modified sequence retains the biologicalactivity of the unmodified sequence. Amino acid substitutions mayinclude the use of non-naturally occurring analogues, for example toincrease blood plasma half-life of a therapeutically administeredpolypeptide.

Natural variants of anti-PRL antibodies are likely to compriseconservative amino acid substitutions. Conservative substitutions may bedefined, for example according to the Table below. Amino acids in thesame block in the second column such as those in the same line in thethird column may be substituted for each other:

Fragments ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

Polypeptides disclosed here and useful as markers also include fragmentsof the above mentioned full length polypeptides and variants thereof,including fragments of the sequences set out in the sequence listings.

Polypeptides also include fragments of the full length sequence of anyof the anti-PRL antibody polypeptides. Fragments may comprise at leastone epitope. Methods of identifying epitopes are well known in the art.Fragments will typically comprise at least 6 amino acids, such as atleast 10, 20, 30, 50 or 100 or more amino acids.

Polypeptide fragments of the anti-PRL antibody proteins and allelic andspecies variants thereof may contain one or more (e.g. 5, 10, 15, or 20)substitutions, deletions or insertions, including conservedsubstitutions. Where substitutions, deletion and/or insertions occur,for example in different species, such as less than 50%, 40% or 20% ofthe amino acid residues depicted in the sequence listings are altered.

Anti-PRL antibody and their fragments, homologues, variants andderivatives, may be made by recombinant means. However, they may also bemade by synthetic means using techniques well known to skilled personssuch as solid phase synthesis. The proteins may also be produced asfusion proteins, for example to aid in extraction and purification.Examples of fusion protein partners include glutathione-S-transferase(GST), 6×His (SEQ ID NO: 40), GAL4 (DNA binding and/or transcriptionalactivation domains) and β-galactosidase. It may also be convenient toinclude a proteolytic cleavage site between the fusion protein partnerand the protein sequence of interest to allow removal of fusion proteinsequences. The fusion protein may be such that it will not hinder thefunction of the protein of interest sequence. Proteins may also beobtained by purification of cell extracts from animal cells.

The anti-PRL antibody polypeptides, variants, homologues, fragments andderivatives disclosed here may be in a substantially isolated form. Itwill be understood that such polypeptides may be mixed with carriers ordiluents which will not interfere with the intended purpose of theprotein and still be regarded as substantially isolated. A anti-PRLantibody variant, homologue, fragment or derivative may also be in asubstantially purified form, in which case it will generally comprisethe protein in a preparation in which more than 90%, e.g. 95%, 98% or99% of the protein in the preparation is a protein.

The anti-PRL antibody polypeptides, variants, homologues, fragments andderivatives disclosed here may be labelled with a revealing label. Therevealing label may be any suitable label which allows the polypeptide,etc to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵I,enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides may be used in diagnostic procedures such asimmunoassays to determine the amount of a polypeptide in a sample.Polypeptides or labelled polypeptides may also be used in serological orcell-mediated immune assays for the detection of immune reactivity tosaid polypeptides in animals and humans using standard protocols.

The anti-PRL antibody polypeptides, variants, homologues, fragments andderivatives disclosed here, optionally labelled, my also be fixed to asolid phase, for example the surface of an immunoassay well or dipstick.Such labelled and/or immobilised polypeptides may be packaged into kitsin a suitable container along with suitable reagents, controls,instructions and the like. Such polypeptides and kits may be used inmethods of detection of antibodies to the polypeptides or their allelicor species variants by immunoassay.

Immunoassay methods are well known in the art and will generallycomprise: (a) providing a polypeptide comprising an epitope bindable byan antibody against said protein; (b) incubating a biological samplewith said polypeptide under conditions which allow for the formation ofan antibody-antigen complex; and (c) determining whetherantibody-antigen complex comprising said polypeptide is formed.

The anti-PRL antibody polypeptides, variants, homologues, fragments andderivatives disclosed here may be used in in vitro or in vivo cellculture systems to study the role of their corresponding genes andhomologues thereof in cell function, including their function indisease. For example, truncated or modified polypeptides may beintroduced into a cell to disrupt the normal functions which occur inthe cell. The polypeptides may be introduced into the cell by in situexpression of the polypeptide from a recombinant expression vector (seebelow). The expression vector optionally carries an inducible promoterto control the expression of the polypeptide.

The use of appropriate host cells, such as insect cells or mammaliancells, is expected to provide for such post-translational modifications(e.g. myristolation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products. Such cellculture systems in which the anti-PRL antibody polypeptides, variants,homologues, fragments and derivatives disclosed here are expressed maybe used in assay systems to identify candidate substances whichinterfere with or enhance the functions of the polypeptides in the cell.

Polynucleotide Sequences

The variable regions, monoclonal antibody sequences and humanisedantibody sequences may comprise polynucleotides. These may comprise DNAor RNA.

They may be single-stranded or double-stranded. They may also bepolynucleotides which include within them synthetic or modifiednucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent document, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides.

Where the polynucleotide is double-stranded, both strands of the duplex,either individually or in combination, are encompassed by the methodsand compositions described here. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included.

Variants, Derivatives and Homologues

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence described in this document include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) nucleotides from or to the sequence. Theresulting sequence may be capable of encoding a polypeptide which hasPRL binding activity as described elsewhere in this document.

As indicated above, with respect to sequence identity, a “homologue” hassuch as at least 5% identity, at least 10% identity, at least 15%identity, at least 20% identity, at least 25% identity, at least 30%identity, at least 35% identity, at least 40% identity, at least 45%identity, at least 50% identity, at least 55% identity, at least 60%identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity, or at least 95% identity to a relevant sequence.

There may be at least 95% identity, such as at least 96% identity, suchas at least 97% identity, such as at least 98% identity, such as atleast 99% identity. Nucleotide homology comparisons may be conducted asdescribed above. A sequence comparison program such as the GCG WisconsinBestfit program described above may be used for this purpose. Thedefault scoring matrix has a match value of 10 for each identicalnucleotide and −9 for each mismatch. The default gap creation penalty is−50 and the default gap extension penalty is −3 for each nucleotide.

Hybridisation

We further describe nucleotide sequences that are capable of hybridisingselectively to any of the sequences presented herein, such as 269, 223and 318 variable region, antibody and humanised antibody or any variant,fragment or derivative thereof, or to the complement of any of theabove. Nucleotide sequences may be at least 15 nucleotides in length,such as at least 20, 30, 40 or 50 nucleotides in length.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction technologies.

Polynucleotides capable of selectively hybridising to the nucleotidesequences presented herein, or to their complement, will be generally atleast 70%, such as at least 80 or 90% and such as at least 95% or 98%homologous to the corresponding nucleotide sequences presented hereinover a region of at least 20, such as at least 25 or 30, for instance atleast 40, 60 or 100 or more contiguous nucleotides.

The term “selectively hybridisable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide isfound to hybridize to the probe at a level significantly abovebackground. The background hybridization may occur because of otherpolynucleotides present, for example, in the cDNA or genomic DNA librarybeing screened. In this event, background implies a level of signalgenerated by interaction between the probe and a non-specific DNA memberof the library which is less than 10 fold, such as less than 100 fold asintense as the specific interaction observed with the target DNA. Theintensity of interaction may be measured, for example, by radiolabellingthe probe, e.g. with ³²P.

Hybridisation conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridisation can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridisation can be used to identifyor detect similar or related polynucleotide sequences.

We disclose nucleotide sequences that can hybridise to a nucleic acid,or a fragment, homologue, variant or derivative thereof, under stringentconditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na₃Citrate pH 7.0}).

Where a polynucleotide is double-stranded, both strands of the duplex,either individually or in combination, are encompassed by the presentdisclosure. Where the polynucleotide is single-stranded, it is to beunderstood that the complementary sequence of that polynucleotide isalso disclosed and encompassed.

Polynucleotides which are not 100% homologous to the sequences disclosedhere but fall within the disclosure can be obtained in a number of ways.Other variants of the sequences described herein may be obtained forexample by probing DNA libraries made from a range of individuals, forexample individuals from different populations. In addition, otherviral/bacterial, or cellular homologues particularly cellular homologuesfound in mammalian cells (e.g. rat, mouse, bovine and primate cells),may be obtained and such homologues and fragments thereof in generalwill be capable of selectively hybridising to the sequences shown in thesequence listing herein. Such sequences may be obtained by probing cDNAlibraries made from or genomic DNA libraries from other animal species,and probing such libraries with probes comprising all or part of thedisclosed sequences under conditions of medium to high stringency.

The polynucleotides described here may be used to produce a primer, e.g.a PCR primer, a primer for an alternative amplification reaction, aprobe e.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, such as at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides as used herein. Fragments may be less than 500, 200,100, 50 or 20 nucleotides in length.

Polynucleotides such as a DNA polynucleotides and probes may be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the sequence which it is desiredto clone, bringing the primers into contact with mRNA or cDNA obtainedfrom an animal or human cell, performing a polymerase chain reactionunder conditions which bring about amplification of the desired region,isolating the amplified fragment (e.g. by purifying the reaction mixtureon an agarose gel) and recovering the amplified DNA. The primers may bedesigned to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be cloned into a suitable cloning vector.

PRL Polypeptides and Nucleic Acids

PRL-1 and PRL-3 polypeptide homologues, variants, derivatives andfragments may be defined similarly, as set out in the previousparagraphs.

Where the context permits, a reference to PRL-1 polypeptide should betaken to include reference to a PRL-1 polypeptide homologue, variant,derivative or fragment. Similarly, a reference to PRL-3 polypeptideshould be taken to include reference to a PRL-3 polypeptide homologue,variant, derivative or fragment.

Similarly, where the context permits, a reference to PRL-1 nucleic acidshould be taken to include reference to a PRL-1 nucleic acid homologue,variant, derivative or fragment. Similarly, a reference to PRL-3polypeptide should be taken to include reference to a PRL-3 nucleic acidhomologue, variant, derivative or fragment.

Anti-PRL Antibody Production

The anti-PRL antibody can be produced by recombinant DNA methods orsynthetic peptide chemical methods that are well known to those ofordinary skill in the art.

By way of example, the anti-PRL antibody may be synthesized bytechniques well known in the art, as exemplified by “Solid Phase PeptideSynthesis: A Practical Approach” E. Atherton and R. C. Sheppard, IRLPress, Oxford England. Similarly, multiple fragments can be synthesizedwhich are subsequently linked together to form larger fragments. Thesesynthetic peptide fragments can also be made with amino acidsubstitutions at specific locations in order to test for activity invitro and in vivo.

The anti-PRL antibody can be synthesized in a standard microchemicalfacility and purity checked with HPLC and mass spectrophotometry.Methods of peptide synthesis, HPLC purification and massspectrophotometry are commonly known to those skilled in these arts.

The anti-PRL antibody may also be expressed under in vitro and in vivoconditions in a transformed host cell into which has been incorporatedthe DNA sequences described here (such as variable sequences) or allelicvariations thereof and which can be used in the prevention and/ortreatment of cancer related diseases.

The term “vector” includes expression vectors and transformationvectors. The term “expression vector” means a construct capable of invivo or in vitro expression. The term “transformation vector” means aconstruct capable of being transferred from one species to another.

Vectors which may be used for expression include recombinant viralvectors, in particular recombinant retroviral vectors (RRV) such aslentiviral vectors, adenoviral vectors including a combination ofretroviral vectors.

The term ‘recombinant retroviral vector” (RRV) refers to a vector withsufficient retroviral genetic information to allow packaging of an RNAgenome, in the presence of packaging components, into a viral particlecapable of infecting a target cell. Infection of the target cellincludes reverse transcription and integration into the target cellgenome. The RRV carries non-viral coding sequences which are to bedelivered by the vector to the target cell. An RRV is incapable ofindependent replication to produce infectious retroviral particleswithin the final target cell. Usually the RRV lacks a functional gag poland/or env gene and/or other genes essential for replication. Vectorswhich may be used include recombinant pox viral vectors such as fowl poxvirus (FPV), entomopox virus, vaccinia virus such as NYVAC, canarypoxvirus, MVA or other non-replicating viral vector systems such as thosedescribed for example in WO9530018.

Pox viruses may be engineered for recombinant gene expression and forthe use as recombinant live vaccines in a dual immunotherapeuticapproach. The principal rationale for using live attenuated viruses,such as viruses, as delivery vehicles and/or vector based vaccinecandidates, stems from their ability to elicit cell mediated immuneresponses. The viral vectors, as outlined above, are capable of beingemployed as delivery vehicles and as vector based vaccine candidatesbecause of the immunogenicity of their constitutive proteins, which actas adjuvants to enhance the immune response, thus rendering a nucleotidesequence of interest (NOI) such as a nucleotide sequence encoding ananti-PRL antibody more immunogenic.

The pox virus vaccination strategies have used recombinant techniques tointroduce NOIs into the genome of the pox virus. If the NOI isintegrated at a site in the viral DNA which is non-essential for thelife cycle of the virus, it is possible for the newly producedrecombinant pox virus to be infectious, that is to say to infect foreigncells and thus to express the integrated NOI. The recombinant pox virusprepared in this way can be used as live vaccines for the prophylaxisand/or treatment of pathologic and infectious disease and/or cancer.

Other requirements for pox viral vector delivery systems include goodimmunogenicity and safety. MVA is a replication-impaired vaccinia strainwith a good safety record. In most cell types and normal human tissue,MVA does not replicate. Limited replication of MVA is observed in a fewtransformed cell types such as BHK21 cells. Carroll et al (1997 Vaccine15: 387-394) have shown that the recombinant MVA is equally as good asconventional recombinant vaccinia vectors at generating a protectiveCD8+T cell response and is an efficacious alternative to the morecommonly used replication competent vaccinia virus. The vaccinia virusstrains derived from MVA, or independently developed strains having thefeatures of MVA which make MVA particularly suitable for use in avaccine, are also suitable for use as a delivery vehicle.

The nucleotide sequence of interest, and of which expression is desired,may operably linked to a transcription unit. The term “transcriptionunit” as described herein are regions of nucleic acid containing codingsequences and the signals for achieving expression of those codingsequences independently of any other coding sequences. Thus, eachtranscription unit generally comprises at least a promoter, an optionalenhancer and a polyadenylation signal. The term “promoter” is used inthe normal sense of the art, e.g. an RNA polymerase binding site. Thepromoter may contain an enhancer element. The term “enhancer” includes aDNA sequence which binds to other protein components of thetranscription initiation complex and thus facilitates the initiation oftranscription directed by its associated promoter. The term “cell”includes any suitable organism. The cell may comprise a mammalian cell,such as a human cell.

The term “transformed cell” means a cell having a modified geneticstructure. For example, as described here, a cell has a modified geneticstructure when a vector such as an expression vector has been introducedinto the cell. The term “organism” includes any suitable organism. Theorganism may comprise a mammal such as a human.

Here the term “transgenic organism” means an organism comprising amodified genetic structure. For example, the organism may have amodified genetic structure if a vector such as an expression vector hasbeen introduced into the organism.

Antibody Expression

We further describe a method comprising transforming a host cell with aor the nucleotide sequences described in this document, such as 269, 223or 318 variable regions, antibody sequences or humanized antibodysequences.

We also provide a method comprising culturing a transformed hostcell—which cell has been transformed with a or the such nucleotidesequences under conditions suitable for the expression of the anti-PRLantibody encoded by said nucleotide sequences.

We further provide a method comprising culturing a transformed hostcell—which cell has been transformed with a or the such nucleotidesequences under conditions suitable for the expression of the anti-PRLantibody encoded by said nucleotide sequences; and then recovering saidanti-PRL antibody from the transformed host cell culture.

Thus, anti-PRL antibody encoding nucleotide sequences, fusion proteinsor functional equivalents thereof, may be used to generate recombinantDNA molecules that direct the expression thereof in appropriate hostcells.

By way of example, anti-PRL antibody may be produced in recombinant E.coli, yeast or mammalian expression systems, and purified with columnchromatography.

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improve tumour tonon-tumour ratios. Fab, Fv, ScFv antibody fragments can all be expressedin and secreted from E. coli, thus allowing the production of largeamounts of the such fragments.

The nucleotide sequences encoding the anti-PRL antibody may be operablylinked to a promoter sequence capable of directing expression of theanti-PRL antibody encoding nucleotide sequences in a suitable host cell.When inserted into the host cell, the transformed host cell may becultured under suitable conditions until sufficient levels of theanti-PRL antibody are achieved after which the cells may be lysed andthe anti-PRL antibody is isolated.

Host cells transformed with the anti-PRL antibody encoding nucleotidesequences may be cultured under conditions suitable for the expressionand recovery of the anti-PRL antibody from cell culture. The proteinproduced by a recombinant cell may be secreted or may be containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining the

Anti-PRL antibody encoding nucleotide sequences can be designed withsignal sequences which direct secretion of the anti-PRL antibodyencoding nucleotide sequences through a particular prokaryotic oreukaryotic cell membrane. Other recombinant constructions may join theanti-PRL antibody encoding nucleotide sequence to a nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-5 3′, seealso the discussion below on vectors containing fusion proteins).

The anti-PRL antibody may also be expressed as a recombinant proteinwith one or more additional polypeptide domains added to facilitateprotein purification. Such purification facilitating domains include,but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals (Porath J (1992) Protein Expr Purif 3-26328 1), protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle, Wash.). The inclusion of a cleavable linker sequence suchas Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and the anti-PRL antibody is useful to facilitatepurification.

The nucleotide sequences described here may be engineered in order toalter a the anti-PRL antibody encoding sequences for a variety ofreasons, including but not limited to alterations which modify thecloning, processing and/or expression of the gene product. For example,mutations may be introduced using techniques which are well known in theart, e.g., site-directed mutagenesis to insert new restriction sites, toalter glycosylation patterns or to change codon preference.

In another embodiment, a or the natural, modified or recombinantanti-PRL antibody encoding nucleotide sequences may be ligated to aheterologous sequence to encode a fusion protein. By way of example,fusion proteins comprising the anti-PRL antibody or an enzymaticallyactive fragment or derivative thereof linked to an affinity tag such asglutathione-S-transferase (GST), biotin, His6 (SEQ ID NO: 40), ac-myctag (see Emrich et al 1993 Biocem Biophys Res Commun 197(1): 21220),hemagglutinin (HA) (as described in Wilson et al (1984 Cell 37 767) or aFLAG epitope (Ford et al 1991 Protein Expr Purif April; 2 (2):95-107).May be produced

The fused recombinant protein may comprise an antigenic coprotein suchas GST, beta-galactosidase or the lipoprotein D from Haemophilllsinfluenzae which are relatively large co-proteins, which solubilise andfacilitate production and purification thereof. Alternatively, the fusedprotein may comprise a carrier protein such as bovine serum albumin(BSA) or keyhole limpet haemocyanin (KLH). In certain embodiments, themarker sequence may comprise a hexa-histidine peptide, as provided inthe pQE vector (Qiagen Inc) and described in Gentz et al (1989 PNAS 86:821-824). Such fusion proteins are readily expressable in yeast culture(as described in Mitchell et al 1993 Yeast 5:715-723) and are easilypurified by affinity chromatography. A fusion protein may also beengineered to contain a cleavage site located between the nucleotidesequence encoding the anti-PRL antibody and the heterologous proteinsequence, so that the anti-PRL antibody may be cleaved and purified awayfrom the heterologous moiety. In another embodiment, an assay for thetarget protein may be conducted using the entire, bound fusion protein.Alternatively, the co-protein may act as an adjuvant in the sense ofproviding a generalised stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Although the presence/absence of marker gene expression suggests thatthe nucleotide sequence for anti-PRL antibody is also present, itspresence and expression should be confirmed. For example, if theanti-PRL antibody encoding nucleotide sequence is inserted within amarker gene sequence, recombinant cells containing the anti-PRL antibodycoding regions may be identified by the absence of the marker genefunction. Alternatively, a marker gene may be placed in tandem with aanti-PRL antibody encoding nucleotide sequence under the control of asingle promoter.

Expression of the marker gene in response to induction or selectionusually indicates expression of the anti-PRL antibody as well.

Additional methods to quantitate the expression of a particular moleculeinclude radiolabeling (Melby P C et al 1993 J Immunol Methods159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36)nucleotides, co amplification of a control nucleic acid. and standardcurves onto which the experimental results are interpolated.

Quantitation of multiple samples may be speeded up by running the assayin an ELISA format where the anti-PRL antibody of interest is presentedin various dilutions and a spectrophotometric or calorimetric responsegives rapid quantitation.

Altered anti-PRL antibody nucleotide sequences which may be made or usedinclude deletions, insertions or substitutions of different nucleotideresidues resulting in a nucleotide sequence that encodes the same or afunctionally equivalent anti-PRL antibody. By way of example, theexpressed anti-PRL antibody may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent anti-PRL antibody. Deliberate aminoacid substitutions may be made on the basis of similarity in polarity,charge. solubility, hydrophobicity, hydrophilicity. and/or theamphipathic nature of the residues as long as the binding affinity ofthe anti-PRL antibody is retained. For example, negatively charged aminoacids include aspartic acid and glutamic acid: positively charged aminoacids include lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values include leucine,isoleucine, valine, glycine, alanine, asparagine, glutamine, serine,threonine, phenylalanine, and tyrosine.

Gene therapy whereby the anti-PRL antibody encoding nucleotide sequencesas described here is regulated in vivo may also be employed. Forexample, expression regulation may be accomplished by administeringcompounds that bind to the anti-PRL antibody encoding nucleotidesequences, or control regions associated with the anti-PRL antibodyencoding nucleotide sequence or its corresponding RNA transcript tomodify the rate of transcription or translation.

By way of example, the anti-PRL antibody encoding nucleotide sequencesdescribed here may be under the expression control of an expressionregulatory element, usually a promoter or a promoter and enhancer. Theenhancer and/or promoter may be preferentially active in a hypoxic orischaemic or low glucose environment, such that the anti-PRL antibodyencoding nucleotide sequences is preferentially expressed in theparticular tissues of interest, such as in the environment of a tumourcell or mass. Thus, any significant biological effect or deleteriouseffect of the anti-PRL antibody encoding nucleotide sequences on theindividual being treated may be reduced or eliminated. The enhancerelement or other elements conferring regulated expression may be presentin multiple copies.

The promoter and/or enhancer may be constitutively efficient, or may betissue or temporally restricted in their activity. Examples of suitabletissue restricted promoters/enhancers are those which are highly activein tumour cells such as a promoter/enhancer from a MUC1 gene, a CEA geneor a STV antigen gene. Examples of temporally restrictedpromoters/enhancers are those which are responsive to ischaemia and/orhypoxia, such as hypoxia response elements or the promoter/enhancer ofagrp78 or agrp94 gene. The alpha fetoprotein (AFP) promoter is also atumour-specific promoter. Another promoter-enhancer combination is ahuman cytomegalovirus (hCMV) major immediate early (MIE)promoter/enhancer combination.

The promoters may be tissue specific. That is, they may be capable ofdriving transcription of a anti-PRL antibody encoding nucleotidesequences in one tissue while remaining largely “silent” in other tissuetypes.

The term “tissue specific” means a promoter which is not restricted inactivity to a single tissue type but which nevertheless showsselectivity in that they may be active in one group of tissues and lessactive or silent in another group. A desirable characteristic of suchpromoters is that they possess a relatively low activity in the absenceof activated hypoxia-regulated enhancer elements, even in the targettissue. One means of achieving this is to use “silencer” elements whichsuppress the activity of a selected promoter in the absence of hypoxia.

The term “hypoxia” means a condition under which a particular organ ortissue receives an inadequate supply of oxygen.

The level of expression of a or the anti-PRL antibody encodingnucleotide sequences under the control of a particular promoter may bemodulated by manipulating the promoter region. For example, differentdomains within a promoter region may possess different gene regulatoryactivities. The roles of these different regions are typically assessedusing vector constructs having different variants of the promoter withspecific regions deleted (that is, deletion analysis). This approach maybe used to identify, for example, the smallest region capable ofconferring tissue specificity or the smallest region conferring hypoxiasensitivity.

A number of tissue specific promoters, described above, may be used. Inmost instances, these promoters may be isolated as convenientrestriction digestion fragments suitable for cloning in a selectedvector. Alternatively, promoter fragments may be isolated using thepolymerase chain reaction. Cloning of the amplified fragments may befacilitated by incorporating restriction sites at the 5′ end of theprimers.

Combination Therapy

The anti-PRL antibodies described here may be used in combination withother compositions and procedures for the treatment of diseases.

By way of example, the anti-PRL antibodies may also be used incombination with conventional treatments of diseases such as cancer. Forexample, a tumor may be treated conventionally with surgery, radiationor chemotherapy combined with a anti-PRL antibody or a anti-PRL antibodymay be subsequently administered to the patient to extend the dormancyof micrometastases and to stabilize any residual primary tumor.

The anti-PRL antibody can be delivered with a therapeutically effectiveagent at the same moment in time and at the same site. Alternatively,the anti-PRL antibody and the therapeutically effective agent may bedelivered at a different time and to a different site. The anti-PRLantibody and the therapeutically effective agent may even be deliveredin the same delivery vehicle for the prevention and/or treatment ofcancer.

Anti-PRL antibodies may be used in combination with cytotoxic agents forthe prevention and/or treatment of angiogenesis and/or cancer. Cytotoxicagents such as ricin, linked to anti-PRL antibodies, anti-PRL antibodiesantisera, anti-PRL antibodies receptor agonists and antagonists providea tool for the destruction of cells that express PRL-1 or PRL-3. Thesecells may be found in many locations, including but not limited to,micrometastases and primary tumours.

Anti-PRL antibodies may be used in combination with a pro-drugactivating enzyme in gene therapy. Instead of or as well as beingselectively expressed in target tissues, the anti-PRL antibody may beused in combination with another molecule, such as a pro-drug activationenzyme or enzymes which have no significant effect or no deleteriouseffect until the individual is treated with one or more pro-drugs uponwhich the enzyme or enzymes act. In the presence of the pro-drugactivation enzyme, active treatment of an individual with theappropriate pro-drug leads to enhanced reduction in tumour growth orsurvival.

A pro-drug activating enzyme may be delivered to a tumour site for thetreatment of a cancer. In each case, a suitable pro-drug is used in thetreatment of the patient in combination with the appropriate pro-drugactivating enzyme. An appropriate pro-drug is administered inconjunction with the vector. Examples of pro-drugs include: etoposidephosphate (with alkaline phosphatase, Senter et al 1988 Proc Natl AcadSci 85: 48424846); 5-fluorocytosine (with cytosine deaminase, Mullen etal 1994 Cancer Res 54: 1503-1506);Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase, Kerret al 1990 Cancer Immunol Immunother 31: 202-206);Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidaseG2); Cephalosporin nitrogen mustard carbamates (with beta-lactamase);SR4233 (with P450 Reductase); Ganciclovir (with HSV thymidine kinase,Borrelli et al 1988 Proc Natl Acad Sci 85: 7572-7576); mustard pro-drugswith nitro reductase (Friedlos et al 1997 J Med Chem 40: 1270-1275) andCyclophosphamide (with P450 Chen et a/1996 Cancer Res 56: 1331-1340).

Examples of pro-drug activation enzymes include a thymidinephosphorylase which activates the 5-fluoro-uracil pro-drugs capcetabineand furtulon; thymidine kinase from Herpes Simplex Virus which activatesganciclovir; a cytochrome P450 which activates a pro-drug such ascyclophosphamide to a DNA damaging agent; and cytosine deaminase whichactivates 5-fluorocytosine. An enzyme of human origin may be used.

Other suitable molecules include those that are of therapeutic and/ordiagnostic application such as, but are not limited to: sequencesencoding cytokines, chemokines, hormones, antibodies, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,enzymes, immune co-stimulatory molecules, immunomodulatory molecules,anti-sense RNA, a transdominant negative mutant of a target protein, atoxin, a conditional toxin, an antigen, a tumour suppressor protein andgrowth factors, membrane proteins, vasoactive proteins and peptides,anti-viral proteins and ribozymes, and derivatives thereof (such as withan associated reporter group). When included, such coding sequences maybe typically operatively linked to a suitable promoter, which may be apromoter driving expression of a ribozyme(s), or a different promoter orpromoters, such as in one or more specific cell types.

The molecules may be proteins which are secreted from the cell.Alternatively the molecules are not secreted and are active within thecell. In either event, the molecules may demonstrate a bystandereffector or a distant bystander effect; that is the production of theexpression product in one cell leading to the killing of additional,related cells, either neighbouring or distant (e.g. metastatic), whichpossess a common phenotype.

Suitable molecules for use in the treatment or prophylaxis of cancerinclude proteins (or nucleic acids encoding proteins) which: destroy thetarget cell (for example a ribosomal toxin), act as: tumour suppressors(such as wild-type p53); activators of anti-tumour immune mechanisms(such as cytokines, co-stimulatory molecules and immunoglobulins);inhibitors of angiogenesis; or which provide enhanced drug sensitivity(such as pro-drug activation enzymes); indirectly stimulate destructionof target cell by natural effector cells (for example, strong antigen tostimulate the immune system or convert a precursor substance to a toxicsubstance which destroys the target cell (for example a prodrugactivating enzyme). Encoded proteins could also destroy bystander tumourcells (for example with secreted antitumour antibody-ribosomal toxinfusion protein), indirectly stimulate destruction of bystander tumourcells (for example cytokines to stimulate the immune system orprocoagulant proteins causing local vascular occlusion) or convert aprecursor substance to a toxic substance which destroys bystander tumourcells (eg an enzyme which activates a prodrug to a diffusible drug).

Antisense transcripts or ribozymes which interfere with expression ofcellular genes for tumour persistence (for example against aberrant myctranscripts in Burkitts lymphoma or against bcr-abl transcripts inchronic myeloid leukemia) may be delivered to enhance cancer cellkilling function or metastasis preventing function of the anti-PRLantibodies. The use of combinations of such molecules is also envisaged.

Examples of hypoxia regulatable therapeutic molecules can be found inPCT/GB95/00322 (WO-A-9521927).

Anti-PRL Antibody Conjugates

The targeting of cells expressing PRL-1 or PRL-3 antigen with theanti-PRL antibodies described here facilitates the development of drugsto modulate the activity of cells expressing PRL-1 or PRL-3.

Different anti-PRL antibodies can be synthesized for use in severalapplications including but not limited to the linkage of a anti-PRLantibody to cytotoxic agents for targeted killing of cells that bind theanti-PRL antibody.

The anti-PRL antibody described here can be coupled to other moleculesusing standard methods. The amino and carboxyl termini of the anti-PRLantibody may be isotopically and nonisotopically labeled with manytechniques, for example radiolabeling using conventional techniques(tyrosine residues—chloramine T. iodogen. lactoperoxidase; lysineresidues—Bolton-Hunter reagent). These coupling techniques are wellknown to those skilled in the art. The coupling technique is chosen onthe basis of the functional groups available on the amino acidsincluding, but not limited to amino, sulfhydral, carboxyl, amide,phenol, and imidazole. Various reagents used to effect these couplingsinclude among others, glutaraldehyde, diazotized benzidine,carbodiimide, and p-benzoquinone.

The anti-PRL antibodies may be chemically coupled to isotopes, enzymes,carrier proteins, cytotoxic agents, fluorescent molecules and othercompounds for a variety of applications. The efficiency of the couplingreaction is determined using different techniques appropriate for thespecific reaction. For example, radiolabeling of an PRL-1 or PRL-3polypeptide with ¹²⁵I is accomplished using chloramine T and Na¹²⁵I ofhigh specific activity. The reaction is terminated with sodiummetabisulfite and the mixture is desalted on disposable columns. Thelabeled antibody is eluted from the column and fractions are collected.Aliquots are removed from each fraction and radioactivity measured in agamma counter. In this manner, the unreacted Na¹²⁵I is separated fromthe labeled PRL-1 or PRL-3 polypeptide. The peptide fractions with thehighest specific radioactivity are stored for subsequent use such asanalysis of the ability to bind to a anti-PRL antibody.

The use of labelled anti-PRL antibodies with short lived isotopesenables visualization quantitation of PRL-1 or PRL-3 binding sites invivo using autoradiographic, or modern radiographic or other membranebinding techniques such as positron emission tomography in order tolocate tumours with anti-PRL antibody binding sites. This applicationprovides important diagnostic and research tools.

In other embodiments, the anti-PRL antibody may be coupled to ascintigraphic radiolabel, a cytotoxic compound or radioisotope, anenzyme for converting a non-toxic prodrug into a cytotoxic drug, acompound for activating the immune system in order to target theresulting conjugate to a colon tumour, or a cell-stimulating compound.Such conjugates have a “binding portion”, which consists of the anti-PRLantibody, and a “functional portion”, which consists of the radiolabel,toxin or enzyme.

The antibody may alternatively be used alone in order simply to blockthe activity of the PRL-1 or PRL-3 antigen, particularly by physicallyinterfering with its binding of another compound.

The binding portion and the functional portion of the conjugate (if alsoa peptide or polypeptide) may be linked together by any of theconventional ways of cross linking polypeptides, such as those generallydescribed in O'Sullivan et of (Anal. Biochem 1979: 100, 100-108). Forexample, one portion may be enriched with thiol groups and the otherportion reacted with a bifunctional agent capable of reacting with thosethiol groups, for example the N-hydroxysuccinimide ester of iodoaceticacid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).Amide and thioetherbonds, for example achieved withm-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stablein vivo than disulphide bonds.

Alternatively, if the binding portion contains carbohydrates, such aswould be the case for an antibody or some antibody fragments, thefunctional portion may be linked via the carbohydrate portion using thelinking technology in EP 0 088 695.

The functional portion of the conjugate may be an enzyme for convertinga non-toxic prodrug into a toxic drug, for example the conjugates ofBagshawe and his colleagues (Bagshawe (1987) Br. 1. Cancer 56, 531;Bagshawe et of (Br. 1. Cancer 1988: 58, 700); WO 88/07378) orcyanide-releasing systems (WO 91/11201).

The functional portion of the anti-PRL antibody conjugate, when theanti-PRL antibody conjugate is used for diagnosis, may comprise orconsist of a radioactive atom for scintigraphic studies, for exampletechnetium 99m (^(99m)Tc) or iodine-123 (¹²³I), or a spin label fornuclear magnetic resonance (nmr) imaging (also known as magneticresonance imaging, mri), such as iodine-123 again, iodine-313,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron.

When used in a compound for selective destruction of the tumour, thefunctional portion of the anti-PRL antibody may comprise a highlyradioactive atom, such as iodine-131, rhenium-186, rhenium-188,yttrium-90 or lead-212, which emits enough energy to destroyneighbouring cells, or a cytotoxic chemical compound such asmethotrexate, adriamicin, vinca alkaliods (vincristine, vinblastine,etoposide), daunorubicin or other intercalating agents.

The radio- or other labels may be incorporated in the anti-PRL antibodyconjugate in known ways. For example, the peptide may be biosynthesisedor may be synthesised by chemical amino acid synthesis using suitableamino acid precursors involving, for example, fluorine-19 in place ofhydrogen. Labels such as ^(99m)Tc, ¹²³I, ¹⁸⁶Rh, ¹⁸⁸Rh and ¹¹¹In can beattached via a cysteine residue in the peptide. Yttrium-90 can beattached via a lysine residue. The IODOGEN method (Fraker et of (1978)Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporateiodine-123. “Monoclonal Antibodies in Immunoscinigraphy” (Chatal, CRCPress 1989) describes other methods in detail.

It may not be necessary for the whole enzyme to be present in theconjugate but, of course, the catalytic portion must be present.So-called “abzymes” may be used, where a anti-PRL antibody is raised toa compound involved in the reaction one wishes to catalyse, usually thereactive intermediate state. The resulting antibody can then function asan enzyme for the reaction.

The conjugate may be purified by size exclusion or affinitychromatography, and tested for dual biological activities. The antigenimmunoreactivity may be measured using an enzyme-linked immunosorbentassay (ELISA) with immobilised antigen and in a live cellradio-immunoassay. An enzyme assay may be used for β-glucosidase using asubstrate which changes in absorbance when the glucose residues arehydrolysed, such as oNPG (o-nitrophenyl-β-D-glucopyranoside), liberating2-nitrophenol which is measured spectrophotometrically at 405 nm.

The stability of the conjugate may be tested in vitro initially byincubating at 37° C. in serum, followed by size exclusion FPLC analysis.Stability in vivo can be tested in the same way in mice by analysing theserum at various times after injection of the conjugate. In addition, itis possible to radiolabel the anti-PRL antibody with ¹²⁵I, and theenzyme with ¹³¹I before conjugation, and to determine thebiodistribution of the conjugate, free anti-PRL antibody and free enzymein an animal, for example a mouse.

Alternatively, the conjugate may be produced as a fusion compound byrecombinant DNA techniques whereby a length of DNA comprises respectiveregions encoding the two portions of the conjugate either adjacent toone another or separated by a region encoding a linker peptide whichdoes not destroy the desired properties of the conjugate.

Conceivably, two of the functional portions of the compound may overlapwholly or partly. The DNA is then expressed in a suitable host in knownways.

Diagnostic Kits

We also disclose diagnostic methods and kits for detection andmeasurement of PRL-1 or PRL-3 in biological fluids and tissues, and forlocalization of PRL-1 or PRL-3 in tissues.

The anti-PRL antibodiess can also be used in a diagnostic method and kitto detect and quantify antibodies capable of binding PRL-1 or PRL-3.These kits may permit detection PRL-1 or PRL-3 which, in certainsituations, may indicate the spread of micrometastases by primarytumours in situ. Patients that have such circulating anti-PRL-1 or PRL-3antibodies may be more likely to develop tumours and cancers, and may bemore likely to have recurrences of cancer after treatments or periods ofremission.

Kits for measurement of PRL-1 or PRL-3 are also contemplated. Theanti-PRL antibodies that possess high titer and specificity can be usedto establish easy to use kits for rapid, reliable, sensitive. andspecific measurement and localization of PRL-1 or PRL-3 in extracts ofplasma, urine, tissues. and in cell culture media.

These assay kits include but are not limited to the followingtechniques; competitive and non-competitive assays, radioimmunoassay,bioluminescence and chemiluminescence assays, fluorometric assays,sandwich assays, immunoradiometric assays. dot blots, enzyme linkedassays including ELISA, microtiter plates, antibody coated strips ordipsticks for rapid monitoring of urine or blood. andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished. Intraassay and interassay variation is established at 20%,50% and 80% points on the standard curves of displacement or activity.

One example of an assay kit commonly used in research and in the clinicis a radioimmunoassay (RIA) kit. After successful radioiodination andpurification of an anti-PRL antibody, the antiserum possessing thehighest titer is added at several dilutions to tubes containing arelatively constant amount of radioactivity, such as 10,000 cpm, in asuitable buffer system. Other tubes contain buffer or pre immune serumto determine the non-specific binding. After incubation at 4° C. for 24hours, protein A is added and the tubes are vortexed, incubated at roomtemperature for 90 minutes, and centrifuged at approximately 2000-2500times g at 4° C. to precipitate the complexes of antiserum bound to thelabeled anti-PRL antibody. The supernatant is removed by aspiration andthe radioactivity in the pellets counted in a gamma counter. Theantiserum dilution that binds approximately 10 to 40% of the labeledanti-PRL antibody after subtraction of the non-specific binding isfurther characterized.

An immunohistochemistry kit may also be used for localization of PRL-1or PRL-3 in tissues and cells. This immunohistochemistry kit providesinstructions, a anti-PRL antibody, and possibly blocking serum andsecondary antiserum linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art.

This immunohistochemistry kit permits localization of PRL-1 or PRL-3 intissue sections and cultured cells using both light and electronmicroscopy. It is used for both research and clinical purposes. Forexample, tumours are biopsied or collected and tissue sections cut witha microtome to examine sites of PRL-1 or PRL-3 production. Suchinformation is useful for diagnostic and possibly therapeutic purposesin the detection and treatment of cancer.

Pharmaceutical Compositions

The anti-PRL antibodies may be effective in treating cancer relateddiseases.

We disclose a method of treating cancer related disease with aneffective amount of a anti-PRL antibody described here. The anti-PRLantibodies may be provided as isolated and substantially purifiedproteins and protein fragments in pharmaceutically acceptablecompositions using formulation methods known to those of ordinary skillin the art.

The anti-PRL antibody may be administered in the form of apharmaceutical composition. Such a pharmaceutical composition mayinclude a therapeutically effective amount of anti-PRL antibody,together with a suitable excipient, diluent or carrier.

The anti-PRL antibody may in particular be introduced into thecirculation of a patient, for example by being injected into a patientvia, e.g., a vein.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

These compositions can be administered by standard routes. These includebut are not limited to: oral, rectal, ophthalmic (including intravitrealor intracameral), nasal, topical (including buccal and sublingual),intrauterine, vaginal or parenteral (including subcutaneous,intraperitoneal, intramuscular, intravenous, intradermaL intracranial,intratracheal, and epidural) transdermal, intraperitoneal. intracranial,intracerebroventricular, intracerebral, intravaginal, intrauterine, orparenteral (e.g., intravenous, intraspinal, subcutaneous orintramuscular) routes.

The anti-PRL antibody formulations may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carriers) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

In addition, the anti-PRL antibodies may be incorporated intobiodegradable polymers allowing for sustained release of the compound,the polymers being implanted in the vicinity of where drug delivery isdesired, for example, at the site of a tumor or implanted so that theanti-PRL antibody is slowly released systemically. The biodegradablepolymers and their use are described, for example, in detail in Brem etof (1. Neurosurg 1991 74:441-446). Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of anti-PRLantibodies through cannulae to the site of interest, such as directlyinto a metastatic growth or into the vascular supply to that tumor.

The anti-PRL antibodies may be linked to cytotoxic agents which areinfused in a manner designed to maximize delivery to the desiredlocation. For example, ricin-linked high affinity anti-PRL antibodiesare delivered through a cannula into vessels supplying the target siteor directly into the target. Such agents are also delivered in acontrolled manner through osmotic pumps coupled to infusion cannulae.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient. It should beunderstood that in addition to the ingredients, particularly mentionedabove, the formulations described here may include other agentsconventional in the art having regard to the type of formulation inquestion.

The anti-PRL antibody conjugates may be administered in any suitableway. usually parenterally, for example intravenously orintraperitoneally, in standard sterile, non-pyrogenic formulations ofdiluents and carriers, for example isotonic saline (when administeredintravenously). Once the anti-PRL antibody conjugate has bound to thetarget cells and been cleared from the bloodstream (if necessary), whichtypically takes a day or so, the pro-drug is administered, usually as asingle infused dose, or the tumour is imaged. If needed, because theanti-PRL antibody conjugate may be immunogenic, cyclosporin or someother immunosuppressant can be administered to provide a longer periodfor treatment but usually this will not be necessary.

The dosage of the anti-PRL antibody described here will depend on thedisease state or condition being treated and other clinical factors suchas weight and condition of the human or animal and the route ofadministration of the compound.

Depending upon the half-life of the anti-PRL antibody in the particularanimal or human, the anti-PRL antibody can be administered betweenseveral times per day to once a week. It is to be understood that themethods and compositions described here have application for both humanand veterinary use. The methods described here contemplate single aswell as multiple administrations, given either simultaneously or over anextended period of time.

The timing between administrations of the anti-PRL antibody conjugateand pro-drug may be optimised in a routine way since tumour/normaltissue ratios of conjugate (at least following intravenous delivery) arehighest after about 4-6 days, whereas at this time the absolute amountof conjugate bound to the tumour, in terms of percent of injected doseper gram, is lower than at earlier times.

Therefore, the optimum interval between administration of the anti-PRLantibody conjugate and the pro-drug will be a compromise between peaktumour concentration of enzyme and the best distribution ratio betweentumour and normal tissues. The dosage of the anti-PRL antibody conjugatewill be chosen by the physician according to the usual criteria. Atleast in the case of methods employing a targeted enzyme such asβ-glucosidase and intravenous amygdalin as the toxic pro-drug, 1 to 50daily doses of 0.1 to 10.0 grams per square meter of body surface area,preferably 1.0-5.0 g/m² are likely to be appropriate. For oral therapy,three doses per day of 0.05 to 10.0 g, preferably 1.0-5.0 g, for one tofifty days may be appropriate. The dosage of the anti-PRL antibodyconjugate will similarly be chosen according to normal criteria,particularly with reference to the type, stage and location of thetumour and the weight of the patient. The duration of treatment willdepend in part upon the rapidity and extent of any immune reaction tothe anti-PRL antibody conjugate.

Diseases

Anti-PRL antibodies described here, for example in the form ofpharmaceutical compositions, may be used in the treatment of cancer.

For the purposes of this document, the term “cancer” can comprise anyone or more of the following: acute lymphocytic leukemia (ALL), acutemyeloid leukemia (AML), adrenocortical cancer, anal cancer, bladdercancer, blood cancer, bone cancer, brain tumor, breast cancer, cancer ofthe female genital system, cancer of the male genital system, centralnervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma,childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloidleukemia (CML), colon and rectal cancer, colon cancer, endometrialcancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladdercancer, gastric cancer, gastrointestinal tract cancer, hairy cellleukemia, head and neck cancer, hepatocellular cancer, Hodgkin'sdisease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer,laryngeal cancer, leukemia, leukemia, liver cancer, lung cancer,malignant fibrous histiocytoma, malignant thymoma, melanoma,mesothelioma, multiple myeloma, myeloma, nasal cavity and paranasalsinus cancer, nasopharyngeal cancer, nervous system cancer,neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer, oropharyngealcancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroidcancer, penile cancer, pharyngeal cancer, pituitary tumor, plasma cellneoplasm, primary CNS lymphoma, prostate cancer, rectal cancer,respiratory system, retinoblastoma, salivary gland cancer, skin cancer,small intestine cancer, soft tissue sarcoma, stomach cancer, stomachcancer, testicular cancer, thyroid cancer, urinary system cancer,uterine sarcoma, vaginal cancer, vascular system, Waldenstrom'smacroglobulinemia and Wilms' tumor.

Anti-PRL antibodies described here, for example in the form ofpharmaceutical compositions, can also be used in the treatment of cancerrelated disorders.

Such disorders include but not limited to: solid tumours; blood borntumours such as leukemias; tumor metastasis; benign tumours, for examplehemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenicgranulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases,for example, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis;plaque neovascularization; telangiectasia; hemophiliac joints;angiofibroma; wound granulation; coronary collaterals; cerebralcollaterals) arteriovenous malformations; ischemic limb angiogenesis;neovascular glaucoma; retrolental fibroplasia; diabeticneovascularisation; helicobacter related diseases, fractures,vasculogenesis, hematopoiesis, ovulation, menstruation and placentation.

EXAMPLES Example 1 Cell Lines: CHO-K1, A2780 and CT26

CHO-K1 cells, A2780 human ovarian cancer cells and CT26 mouse coloncancer cells are purchased from ATCC (Manassas, Va.).

Example 2 Generation of CHO Cell Pools Stably Expressing EGFP-PRL-3 orEGFP-PRL-1

CHO cell pools stably expressing EGFP-PRL-3 or EGFP-PRL-1 are generatedas described in a previous study⁸. Briefly, the cells are cultured inRPMI 1640 medium supplemented with 10% fetal bovine serum and selectedin 1 mg/ml G418 for 20-30 days. The cells (10⁶ cells/ml) are thensubjected to EGFP sorting by FACS Vantage, SE mode (Becton Dickinson)and re-grown in culture to establish stable cell pools.

Example 3 Generation of AT-3 Cells Stably Expressing EGFP-PRL-3 or AT-1Cells Stably Expressing EGFP-PRL-1

To obtain EGFP-PRL-3 or EGFP-PRL-1 tumours, 8-week old nude mice(Jackson Labs, USA) are each injected via the tail vein with EGFP-PRL-3-or EGFP-PRL-1-expressing cells (5×10⁵). Mice are sacrificed at 3 weeksafter the tail vein injection. Lungs carrying EGFP-PRL-3 or EGFP-PRL-1tumours are removed. EGFP-PRL-tumours are dissected out individuallyunder the fluorescent microscope (Zeiss, M2 Bio Quad). To generate celllines derived from these tumours, each EGFP-PRL-tumour is washed twicein PBS under sterile conditions. The tumour is cut into tiny pieces andcultured at 37° C. with 5% CO₂ with RPMI 1640, 10% FBS and 1%antibiotics (Sigma). AT-3 tumour cell line is derived from theEGFP-PRL-3 tumour; while AT-1 tumour cell line is derived from theEGFP-PRL-1 tumour. The cells are trypsinized and split at a ratio of 1:3into new dishes. The tumour cell lines homogeneously expressedEGFP-PRL-3 or EGFP-PRL-1 are confirmed by indirect immunofluorescence.

Example 4 Generation of Specific PRL-3 mAbs Clones 223, 318 and PRL-1mAb Clone 269

These antibodies are generated as follows (see also reference 19):Hybridomas are generated using ClonaCell-HY Hybridoma Cloning Kit fromStemcell Technologies, Inc. (Vancouver, British Columbia, Canada). Theprocedures are followed according to the manufacturer's directions.Briefly, the following are done: (a) immunization of BALB/c mice withGST-mouse whole PRL-1, or PRL-3 fusion protein, respectively; (b) growthof BALB/c parental myeloma cells SP2/0; (c) preparation of BALB/c micefor spleenocytes from immunized mice; (d) fusion of spleenocytes withSP2/0 cells; and (e) selection and characterizations of the hybridomaclones.

Two control ascites: 1. Ascitic fluid derived from hybridoma 6A7Magainst human glycophorin A (obtained from ATCC). 2. Ascitic fluidagainst to GS28 Golgi complex marker.

Example 5 Experimental Metastasis Assay

Reference is made to reference 20. All animal studies have been approvedby the Review Board of the Institute of Molecular and Cell Biology,Singapore. We follow the policies from the Animal Facility Center of TheAgency for Science, Technology and Research (A* STAR),Singapore^(5,8,19). Nude mice are injected with one million cancer cellsvia their tail vein on day 1. The treated mice are administrated withPRL-mAbs via tail vein twice a week.

Example 6 Western Blot Analysis

Detailed steps are described in reference 19.

Example 7 Confocal Microscopy and Analysis of EGFP-PRL-3-ExpressingCancer Cells

AT-3, AT-1, or parental CHO cells are grown on cover slips and washedonce with PBSCM (PBS containing 1 mM MgCl₂ and 1 mM CaCl₂). Cells arethen fixed in 2.7% paraformaldehyde for 20 min at room temperature (RT,24° C.). After two more washes with PBSCM, cells are permeabilized for15 min with 0.12% Saponin in PBSCM (this step is omitted fornon-permeabilized cells) and incubated with anti-PRL-3 mAb. The cellsare washed gently three times with PBSCM and incubated with anti-mouseIgG conjugated with Texas Red (Sigma) for 4 hours at RT. AT-3 or AT-1cells are directly visualized with fluorescence microscopy in green. Toexamine antibodies taken up in live parental CHO cells, cells are firstincubated with mouse anti-GS28 antibody at 4° C. for 2 h. The cells arewashed gently three times with PBSCM and then fixed in 0.6%paraformaldehyde for 20 min at room temperature (RT, 24° C.). The cellsare incubated with anti-mouse IgG conjugated with FITC (Sigma) for 4hours at RT. To-pro-3 iodide is used to stain the DNA of every parentalCHO cell in blue. Confocal imaging is performed with a Zeiss LSM 510image Browser.

Example 8 Immunohistochemistry (IHC)

Using monoclonal mouse PRL-3 (clone 223 or 318) or mouse PRL-1 (clone269) antibodies (1:300 dilution) and VECTASTAIN ABC kit-PeroxidasesRabbit IgG PK-4001 (Orton Southgate, Peterborough, England) to performIHC experiments, we investigated PRL-3 and PRL-1 protein expressions onhuman colon and breast cancer specimens from Cybrdi (Frederick, Md.).The human low and high density multiple malignant tumour arrays(TS42040704) and (TS43040303) are purchased from BioGenex (San Ramon,Calif.). The formalin-fixed, paraffin-embedded slides are baked at 54°C. for 10 min and then de-waxed in fresh xylene for 5 min (2×). Theslides are subjected to rehydration with sequential 100%, 95%, 80%, and75% Ethanol, PBS (2 min for each change), then in 0.01M sodium citratepH 6.0 buffer, followed by antigen retrieval 2100-Retriever Pick CellLaboratories (Amsterdam, North Holland, 1098SM, NL) for 15 min. Theslides are cooled for 4 h in the cooker. The slides are washed threetimes with PBS (5 min each) and transferred into PBS with 0.6% H₂O₂ inthe dark for 20 min. The slides are washed in PBS several times andtreated in PBS-0.2% Tween 20 for 20 min at 24° C. The blocking step andantibodies incubations are performed according to the manufacturer'sinstructions.

Example 9 An Animal Model Allows Rapid Formation of AggressiveMetastatic Lung Tumours

This Example describes the generation of PRL-over expressing tumours inmice and provision of an animal model for future PRL-cancer therapy.

Reference is made to FIG. 1.

Firstly, nude mice are used as an in vivo “cell sorter” to select cellswith high metastatic activities. 1×10⁶ EGFP-PRL-3- or EGFP-1-expressingCHO cells are injected into the circulation of nude mice via the tailvein. Dozens of metastatic tumours or foci are formed in the lung ofeach nude mouse at three weeks post-injection.

Secondly, a single such lung metastatic EGFP-PRL-tumour is thendissected out and minced in culture dishes to establish a moreaggressive and homogeneous EGFP-PRL-3 expressing metastatic cell linenamed AT-3 or EGFP-PRL-1 expressing tumour cell line named AT-1.

Thirdly, 1×10⁶ of AT3 or AT1 cells are injected into nude mice again viathe tail vein. Fourthly, we divided the mice into untreated or treatedgroups with different antibodies administrated via tail vein injectionon days 3, 6, and 9-post inoculation of the tumour cells.

An animal model in which PRL-3 or PRL-1 over-expressing cells rapidlyformed metastatic tumours is designed. With such aggressive metastatictumours as background, one should be able to observe any suppression oftumourigenicity if treatment with anti-PRL-mAbs is effective.

Our animal model recapitulates the aggressive metastatic activities ofPRL-expressing cells and is useful in dissecting metastatic eventsoccurring after invasion or intravasation.

Example 10 PRL-3 mAb or PRL-1 mAb Blocks the Formation of EGFP-PRL-3 orEGFP-PRL-1 Metastatic Lung Tumours with ˜90% Efficacy in Mice

Four groups of control mice (FIG. 2A, a, untreated; b, PBS-treated; c &d, two unrelated antibodies) showed massive and widespread EGFP-PRL-3metastatic tumours (˜140-150 loci) in their lungs on day15-post-injection of cells.

Strikingly, the other four groups of mice that received three doses ofPRL-3 mAbs in the form of either purified IgG or unpurified asciticfluid from hybridoma clone 223 (FIG. 2A e and g) or clone 318 (FIG. 2A fand h) showed a dramatic reduction in EGFP-PRL-3-expressing tumours(−15-20 loci) in their lungs.

Similarly, the PRL-1 mAb significantly reduced the formation ofmetastatic tumours derived from the inoculation of EGFP-PRL-1-expressingAT-1 cells (FIG. 2B, lane 9). Overall, mice (n=40) treated with PRLmonoclonal antibodies showed inhibition of metastatic lung tumours by˜90% compared to control mice (n=40). PRL-3 mRNA (but not protein) hasbeen detected in the heart⁵.

In this regard, it is worth emphasizing that animals treated with thePRL-3 mAb did not exhibit noticeable cardiotoxicity or other undesirableside effects.

Example 11 PRL-1 mAb Specifically Blocks the Formation of PRL-1 (but notPRL-3) Metastatic Tumours while PRL-3 mAb Specifically Blocks theFormation of PRL-3 (but not PRL-1) Metastatic Tumours

This Example demonstrates that the effects of the antibodies arespecific.

Reference is made to FIG. 3.

We show that formation of lung metastatic tumours by PRL-1- andPRL-3-expressing cells is not blocked by mock-PBS treatment (a, e) butis effectively blocked by rabbit PRL antibodies (b, f) as the rabbitantibodies react with all three PRLs (PRL-1, PRL-2 and PRL-3).

We show PRL-1 mAb blocks the formation of lung metastatic tumours inwhich PRL-1 is overexpressed (c) but not PRL-3 is overexpressed (g).

Similarly, PRL-3 mAb inhibits (reducing tumour numbers) the formation oflung metastatic tumours in which PRL-3 is overexpressed (h) but notPRL-1 is overexpressed (d).

Example 12 PRL-3 mAbs Effectively Inhibit the Formation of MetastaticTumours by A2780 Cells that Express Endogenous PRL-3 but not CT26 Cellsthat do not Express Endogenous PRL-3

The dramatic efficacy of PRL-mAbs therapies depends on targeting cellsthat have high levels of expression of the EGFP-PRL proteins so far.

We then performed a crucial experiment to assess if the antibodies couldblock metastasis of cancer cells that naturally express PRL-3 protein.Candidate cancer cell lines that are ideal to be used in this experimentshould have two properties: 1. naturally expressed PRL-3 protein. 2. beable to cause metastatic tumour formation in mice rapidly.

We found A2780 human ovarian cancer cell line has these two propertiesand confirmed that A2780 cells express PRL-3 protein naturally (FIG. 4A,lane 2), which is reported previously⁹.

Meanwhile, we identified a mouse colon cancer cell line CT26 that doesnot express PRL-3 protein (FIG. 4A, lane 1). CT26 cancer cell line isselected as a negative control for this experiment as it has strongmetastatic activity in lungs of nude mice at 2-week post inoculation ofthe cancer cells (FIG. 4B).

Expectedly, no difference is found between treated (FIG. 4B, a) anduntreated (FIG. 4B, b) groups of mice receiving CT26 cells, which allshow pathologic appearance in weight loss.

Rapid formation of aggressive lung metastatic tumours by CT26 cells isnot inhibited by PRL-3 mAbs (FIG. 4B, bottom a) as compared with lungs(FIG. 4B, bottom b) from untreated mice, suggesting that the PRL-mAbs donot inhibit the formation of lung metastatic tumours in which PRL-3phosphatase is not expressed naturally.

In contrast, significant differences are found between treated anduntreated mice at 1-month post-inoculation of A2780 human cancer cellsthat express endogenous PRL-3 protein. Pathologic appearances (unhealthyand skinny) as well as multiple tumours formed by A2780 PRL-3positive-cells are observed only in untreated mice (FIG. 4C, R-side) butnot in treated mice (FIG. 4C, L-side). The treated mice remain healthyfor a prolonged period of time.

These results suggest that PRL-3 mAbs are able to block metastatictumour formation of cancer cells naturally expressing PRL-3 but has noeffect on cancer cells that do not express endogenous PRL-3.

Example 13 PRL-3- or PRL-1-Expressing Cells Taking Up their RespectivePRL-Antibodies

The precise mechanism by which anti-PRL antibodies inhibit tumourformation needs to be further investigated. To determine whether tumourcells can take up PRL mAbs, we examined PRL-3 mAb staining using anindirect immunofluorescence assay in non-permeabilized AT-3 cellsover-expressing EGFP-PRL-3.

Some non-permeabilized AT-3 cells appeared fully stained with theanti-PRL-3 mAb (FIG. 5A, white arrows indicated in b & e panels). 40% ofthese cells are partially stained in red, but >50% of the cells arecompletely unlabeled (FIG. 5A, b, e) compared to permeabilized AT-3cells showing 100% staining in red with anti-PRL-3 mAb (FIG. 5A, h).

The data indicate that large PRL mAbs are still able to either partiallyor completely penetrate into non-permeabilized cancer cells.

Analogous results are obtained using an anti-PRL-1 mAb (clone #269) withCHO cells that over-express EGFP-PRL-1 (data not shown).

To further confirm the antibody can access into cells internally, weselect an antibody against to Golgi apparatus marker (GS28) and showthat the GS28 mAb can penetrate into CHO live cells in culture (FIG. 5Ba-c). Furthermore, the majority (60-70%) of live cells that areserum-starved overnight shows higher efficacy in taking up mouseanti-GS28 by unknown mechanisms (shown in green FIG. 5B d-f).

To investigate if the up-take of the antibody is a general cellphenomenon or not, three PRL-unrelated antibodies are tested on twoother cell lines. These are mouse anti-GS28 and rabbit anti-PTENantibodies on non-permeabilized human mammary epithelial cells(MCF-10A); and mouse anti-GS28 and rabbit anti-p53 on human breastcancer cells (MCF-7).

Again, we found that regardless of whether the antibody source is rabbitor mouse, a fraction of non-permeabilized cells (about 10%) showedefficient uptake of both antibodies in the same cells (FIG. 6A, B). WhenMCF-7 cells are serum-starved overnight, the non-permeabilized MCF-7cells are able to take up both GS28 and p53 antibodies with highlyefficiency (FIG. 6C).

The indirect immunofluorescence double staining reveals a generalphenomenon of antibody uptake in normal and cancer cells. Antibodies canbe taken up by non-permeabilized cells, and this is enhanced by serumstarvation of cells. The results suggest that intracellular proteins mayalso be targeted using monoclonal antibody therapies.

Example 14 Over-Expression of PRL-3 and PRL-1 is Associated with aVariety of Metastatic Cancers

To evaluate the clinical and prognostic significance of PRL-mAbs aspotential drugs against PRL-3- or PRL-1-related cancers, we sought toadditionally assess a spectrum of known PRL-mediated cancers whichinclude colon, breast, lung, brain, ovary, melanoma, and gastriccancers^(3-12,19).

Using a PRL-3 mAb to perform immunohistochemistry on human multiplecancer tissue arrays, we found that PRL-3 protein is up-regulated in 15%(26/158 cases) of colon cancers, 16.5% (19/96 cases) of breast cancers,and 15% (2/13 cases) of esophagus cancers.

Over-expression of PRL-3 is closely associated with squamous cellcarcinoma in lung, penis, and cervix cancers. Selected samples are shownin FIG. 7A.

PRL-1 protein is over-expressed in 6.2% (8/128 cases) of colon cancers;in 20% (5/20 cases) of brain cancers, and in 7.6% (1/13 cases) ofesophagus cancers (FIG. 7B). In these cancer samples, PRL-positivesignals are mainly localized at the plasma membrane and the cytoplasm.

Example 15 Discussion (Example 1 to Example 14)

Metastasis is the most fearsome aspect of cancer. We and others haveshown that over-expression of PRL-3 and PRL-1 is associated with avariety of human cancers^(3-12,19). In this study, we furtherdemonstrate that PRL-3 protein is up-regulated in colon, breast andesophagus cancers. Over-expression of PRL-3 is closely associated withsquamous cell carcinoma in lung, penis, and cervix cancers. PRL-1protein is also over-expressed in colon, brain and esophagus cancers.

Targeting cancer cells that over-express intracellular PRL to preventcancer metastasis by exogenous reagents is a challenging task.Monoclonal antibodies (mAbs) constitute the most rapidly growing classof human therapeutics and are proven agents for recognizing anddestroying malignant cells. In order to block PRL-mediated cancermetastases, we need to ablate PRL-expressing cancer cells to preventthem from further spreading. We attempt to approach this challenging aimwith monoclonal antibody therapy in mice. Using an experimentalmetastasis assay in which cultured PRL-tumour cells are directlyintroduced into each mouse via its tail, we examined in vivo growth andtumour formation of cancer cells. There are four major steps in theprocess of cancer metastasis. Firstly, cancer cells have to enhancemigratory ability in order to escape and dissociate from primary tumour.Secondly, cancer cells need to enter and survive in the bloodcirculation (intravasation). Thirdly, cancer cells ought to get out fromthe blood vessels (extravasation) in order to land on a new organ.Fourthly, cancer cells (seeds) need to survive in the distant organs(soils) to grow up into metastatic tumours. We started our metastaticassay at the step of intravasation by injecting one millionEGFP-PRL-cancer cells directly into blood vessel via the tail vein (FIG.1). The process of cancer metastasis is a long and difficult journey;only about 0.01% (˜100 tumours out of 1×10⁶ cancer cells) of cancercells is estimated to reach their final destination successfully. By theend of the experiment, we observed about 100-150 metastatic lung tumoursin untreated mice (FIG. 2A, a-d) and about 10-15 metastatic lung tumoursin mice treated with PRL-specific mAbs (FIG. 2A, e-h). Our studiesrepresent the first examples of effectively (˜90%) blocking experimentalmetastasis in mice by using mAbs against their respective phosphatasesdespite their intracellular localization. In addition, we showed thatPRL-mAb is specific to its own antigen. PRL-1 mAb specifically blocksthe formation of PRL-1 but not PRL-3 metastatic tumours; while PRL-3 mAbspecifically blocks the formation of PRL-3 but not PRL-1 metastatictumours (FIG. 3). Furthermore, we demonstrated that the PRL-3 mAbs donot block tumour formation by CT26 mouse colon cancer cells in which theendogenous PRL-3 phosphatase is not expressed (FIG. 4A, B).Significantly, we show that PRL-3 mAbs effectively block the formationof metastatic tumours by a human ovarian cancer cell line A2780 thatexpresses endogenous PRL-3 protein. The effective inhibition ofmetastatic tumour formation by PRL-3 positive naturally-occurring humancancer cells is important as it indicates that PRL-3 mAb or itshumanized antibodies may be candidates to treat human cancers associatedwith PRL-3 overexpression. Although the comparison of PRL-3 antibody'sinhibitory effect on A2780 human ovarian cancer cells with thenon-inhibitory effect on mouse CT26 colon cancer cells indicates theantibody is targeting cancer cells endogenously expressing PRL-3, futurestudies employing isogenic cancer cells differing only in the expressionlevels of PRL-3 are required to convincingly demonstrate this point.

There are a number of possible mechanisms responsible for PRL-3antibody-mediated inhibition of tumour formation in the experimentalmetastasis assay

Firstly, the antibody may potentially enter into PRL-3 expressing cellsto target intracellular PRL-3 and neutralize its function. The uptake ofantibody against PRL-3 by a fraction of PRL-3 expressing cancer cells inculture and the enhancement of antibody uptake upon serum-starvationseem to support this mode of action. Secondly, a small fraction of PRL-3may be externalized and displayed on the surface of the PRL-3 expressingcells. Binding of antibody to surface-exposed PRL-3 may trigger immuneresponses such as complement-mediated cytotoxicity (CDC),antibody-dependent cellular cytotoxicity (ADCC), and/orcomplement-dependent cellular cytotoxicity (CDCC) to destroy the cancercells. Thirdly, intracellular PRL-3 could be proteolytically processedand antigenic fragments may be presented on the cell surface by class Imajor histocompatibility antigen so that the cancer cells become targetsof cytotoxic T cells.

To address the first possibility for how these PRL-mAbs inhibit cancermetastasis driven by their respective intracellular antigens, we provideevidence that the uptake by PRL-3- or PRL-1-expressing cells of theirrespective PRL-antibodies might be a general phenomenon as we also foundthat a PRL-unrelated antibody to the Golgi marker-GS28 can penetrateinto live CHO cells in culture and the antibody uptake is enhanced byserum starvation of the cells (FIG. 5B).

An important question is whether the antibody penetration occursspecifically in cancer cells or in untransformed cell types as well, andif other classes of antibody can also enter cells. Three PRL-unrelatedantibodies are tested on two other cell lines. These are mouse anti-GS28and rabbit anti-PTEN antibodies on non-permeabilized human mammaryepithelial cells (MCF-10A); and mouse anti-GS28 and rabbit anti-p53 onhuman breast cancer cells (MCF-7).

Again, we found that regardless of whether the antibody source is rabbitor mouse, a fraction of non-permeabilized cells (about 10%) showedefficient uptake of both antibodies in the same cells (FIG. 6A, B). WhenMCF-7 cells are serum-starved overnight, the non-permeabilized MCF-7cells are able to take up both GS28 and p53 antibodies with highefficiency (about 70% FIG. 6C).

Serum-starvation is used to arrest cells at G1 and G0 phases²¹. It ispossible that particular stages of the cell cycle can contribute to theabilities of cells to take up the antibodies. In vivo, cancer cells areunder hypoxic stress and serum deprivation, conditions that mightenhance the abilities of cancer cells to take up antibodies.

The findings suggest a hitherto unrecognized general phenomenon thatcells are able to take up antibodies to neutralize intracellularantigens. In particular, we have demonstrated that PRL-3 and PRL-1antibodies specifically target their respective intracellular proteinsand ablate tumour formation with no detectable side effects in theseanimals. Although the specific steps of tumour formation in theexperimental metastasis assay inhibited by PRL-3 antibody remain to bedefined by future studies, our results indicate that single cells (ormicro-metastases consisting of cluster cells) seeded in the lung orother secondary tissues are likely the targets of PRL-3 antibody asreflected by the dramatic reduction in the numbers of tumour nodules.Since the seeding of cancer cells in secondary tissues and progressionof micrometastases into macrometastases are major limiting steps incancer spread, targeting these stages with PRL-3 antibodies is ofpotential clinical relevance in preventing cancer metastasis.

Here we provide evidence to demonstrate the PRL-3 mAbs are correctlytargeted to tumours with endogenous expression of PRL-3. Our datafinally suggest that intracellular proteins may also be targeted usingmonoclonal antibody therapies to ablate metastatic tumour formation. Wepropose that cancer researchers consider reevaluating a wide spectrum ofintracellular oncoproteins as possible targets of mAbs for anticancertherapy.

Example 16 Generation of Specific PRL-3 and PRL-1 Mouse/Human ChimericmAbs (Clone #318, 269)

For PRL-3 chimeric mAb, the total RNA is extracted from 6×10⁶ hybridomacells (clone#318) using RNeasy Mini Kit (QIAGEN, cat#74104). DNAse isused during RNA extraction. The RNAs are then reverse-transcribed intocDNA using SuperScript II RNase H (Invitrogen, Cat 18064-014). Theresulting total cDNAs are used as templates to generate ‘universalvariable region’ using Ig-Prime Kits (Novagen, cat#69831-3) for PCR (95°C., 54° C., 72° C.) with 30 cycles. The PCR fragment is cloned intoPCRII-TOPO-Vector with TA cloning kit (Invitrogen, part#45-0640). ThePCR fragment is cut with Mfe1 and XhoI then inserted into respectivesites of a human IgG1 constant region expression vector-pCMV-human IgG1(18) to join mouse variable region of heavy chain (clone #318) withhuman IgG1 constant region. Similar PCR procedures are performed formouse variable region of light chain with ends containing restrictionsites for ApaL1 and Pst 1 are used to PCR the mouse variable light chain(clone #318). The PCR fragment is cut with ApaL1 and Pst I and theninserted into respective sites of a human IgG1 constant regionexpression vector containing variable region of heavy chain of clone#318. The complete construct is transiently transfected into 293T cellswhich are cultured with ultra-low IgG FBS (Gibco, 16250-078). Thechimeric mAb is harvested from the culture supernatant and concentratedup to 40 times with centrifugal filter devices (Millipore,cat#UFC900596). The chimeric mAb is tested for its specificity byindirect immunofluorescence (IF) and Western blot analysis. To generatePCR fragment for PRL-1 (clone #269) variable region of light chain,similarly, mRNAs is extracted from hybridoma cells #269 and the mRNAsare then reverse-transcribed into cDNAs that are used to retrieve thecoding sequence of the variable region of heavy and light chains. Togenerate PCR fragment for PRL-1 variable region of light chain, similarprocedures are carried as mentioned above.

Example 17 Generation of DLD-1-EGFP-PRL-3 Tumour Cell Line

DLD-1 colon carcinoma cells from ATCC CCL-221 (Mannassas, Va.).EGFP-PRL-3 expression construct is transfected into DLD-1 cells usingLipofectamine 2000 from Invitrogen (Carlsbad, Calif.). To obtainEGFP-PRL-3 tumours, 8-week old nude mice (Jackson Labs, USA) are eachinjected into the hips of nude mice to form xenograft tumour. Mice aresacrificed at 3 weeks after cancer cell inoculation. Tumours are removedand examined under the fluorescent microscope (Zeiss, M2 Bio Quad). Togenerate DLD-1-EGFP-PRL-3 tumour cell line, EGFP-tumour is washed twicein PBS under sterile conditions; cut into tiny pieces and cultured at37° C. with 5% CO₂ with RPMI 1640, 10% FBS and 1% antibiotics (Sigma).The cells are trypsinized and split at a ratio of 1:3 into new dishes.The tumour cell lines homogeneously expressed EGFP-PRL-3 or EGFP-PRL-1are confirmed by indirect immunofluorescence.

Example 18 Cell Lines: HCT116 (CCL-247), DLD-1 (CCL-221), B16F0(CRL-6475), B16F10 (CRL-6322), A2780

HCT116 (CCL-247) is a human colorectal carcinoma cell line. DLD-1(CCL-221) is a human colorectal adenocarcinoma cell line. B16F0(CRL-6475) and B16F10 (CRL-6322) are two mouse melanoma cell lines. Allfour cell lines are purchased from ATCC. A2780 is a human ovarian cancercell line and is purchased from ECACC (Cat#93112519 UK).

Example 19 Experimental Animals

Reference is made to document A19. All animal studies have been approvedby our Institute's Review Board. We follow the policies from the AnimalFacility Center of The Agency for Science, Technology and Research (A*STAR), Singapore. Eight-week nude mice (Jackson Labs, USA) are used.1×10⁶ cancer cells are injected into the circulation of nude mice viathe tail vein on day 1. Either chimeric mAb for treated mice or PBS foruntreated mice is administrated into tail vein on day 3.

Example 20 Generation of Specific PRL-3 Mouse/Human Chimeric mAb (Clone#318)

We are encouraged by the fact that PRL-1 and PRL-3 mouse mAbs couldspecifically target their respective intracellular PRL phosphatases andinhibit cancer metastases in experimental animals (reference A16). In anattempt to bring the laboratory work to clinic, we engineered amouse/human chimeric mAb against PRL-3 to reduce the potentialantigenicity of the mouse mAb in human. The PRL-3 chimeric mAb issuccessfully developed in which the constant domains of the human IgGmolecule (reference A18) are combined with the mouse variable regions(heavy and light chains) of PRL-3 mAb clone#318 by transgenic fusion ofthe immunoglobulin genes (FIG. 8A) that is performed by a recombinantDNA technology. The expression construct is transfected into HumanEmbryonic Kidney cells expressing simian virus 40 T antigen (293T) cellsto produce the chimeric PRL-3 mAb that is then harvested from theculture medium and further concentrated by 40 times.

Example 21 Generation of Specific PRL-1 Mouse-Human Chimeric mAb (Clone#269)

We carried out the similar strategy to generate PRL-1 mouse variableregions of heavy and light chains, and then are respectively interestedinto the constant domains of the human IgG expression vector (referenceA18) to generate chimeric PRL-1 mAb.

Example 22 The PRL-3 and PRL-1 Chimeric mAbs Specifically Target totheir Antigens

The PRL-3 and PRL-1 chimeric mAbs are confirmed for their antigenspecificities by performing indirect immuofluorescence (IF) (FIG. 8B)and western blot analyses (FIG. 8C and FIG. 8D). The data show that thePRL-3 chimeric mAb recognizes only PRL-3 but not PRL-1 and -2 proteins;while the PRL-1 chimeric mAb binds only to PRL-1 but not to PRL-2 and -3proteins.

Example 23 PRL-3 Chimeric Antibody Effectively Inhibits the Formation ofMetastatic Tumours by A2780 Cells and HCT116 that Express EndogenousPRL-3; but not DLD-1 Cells that do not Express Endogenous PRL-3

A crucial experiment is performed to assess if the PRL-3 chimeric mAbcould target and block the formation of metastatic tumours derived fromcancer cells that naturally express PRL-3 protein. Dozens of cancercells are screened for PRL-3 expression by western blot analysis. Thecandidate cancer cell lines that are ideal to be used in this experimentshould have two properties: 1. naturally expressed PRL-3 protein. 2. beable to cause metastatic tumour formation rapidly. We found A2780 humanovarian cancer cell line and HCT116 human colorectal cancer cell linehave these two properties and confirmed that A2780 cells and HCT116cells express PRL-3 protein naturally (FIG. 9A lane 1, 2). A2780 cellsare reported previously as PRL-3 positive cell line (reference A4).Remarkably, significant differences are found between PRL-3 chimeric mAbtreated and untreated mice at 1-month post-inoculation of HCT116 (n=5)or A2780 (n=8) cells. Pathologic appearances (unhealthy and skinny) areobserved only in untreated mice (FIG. 9B and FIG. 9C, L-side) but not intreated mice (FIG. 9B and FIG. 9C, R-side). The treated mice remainhealthy for a prolonged period of time. These results suggest that PRL-3chimeric mAb is able to block metastatic tumour formation of cancercells naturally expressing PRL-3. In contrast, we identified a humancolon cancer cell line DLD-1 that does not express PRL-3 protein (FIG.9A, lane 3). The DLD-1 cells are serviced as a negative control for thisexperiment. Expectedly, no difference is found between treated (FIG. 9D,L-side) and untreated (FIG. 9D, R-side) groups (n=5) of mice receivingDLD-1 cells, which all show healthy appearance, at 3.5-monthpost-inoculation of DLD-1 cells. Strikingly, DLD-1 cells engineered tooverexpress EGFP-PRL-3 could cause pathological phenotype in mice at2-month post-inoculation of the cells. Multiple micro-metastatic tumoursare found in the lungs of nude mice carrying these exogenous PRL-3expressing cancer cells. The EGFP-PRL-3 positive cells are also found inthe blood smear of the untreated mice (FIG. 10A n=5) at this time;suggesting that PRL-3 could prolong cell survival in the blood stream.In contrast, mice received PRL-3 chimeric mAb treatment showedsignificantly difference in their sizes and healthy appearance for aprolonged period of time. Micro-metastatic tumours are not found in thelungs of treated nude mice carrying these exogenous PRL-3 expressingcancer cells. The EGFP-PRL-3 positive cells are less found in the bloodsmear of the treated mice (FIG. 10B n=5). The results suggest that PRL-3chimeric mAb is able to block metastatic tumour formation of cancercells that either express endogenous PRL-3 naturally or expressengineered PRL-3 exogenously. Importantly, the antibody has no effect oncancer cells that do not express endogenous PRL-3.

Example 24 PRL-3 Chimeric Antibody Effectively Inhibits the Formation ofMetastatic Tumours by B16F0 Cells that Express Endogenous PRL-3; but notB16F10 Cells that do not Express Endogenous PRL-3

We had demonstrated that the antibody could block metastatic tumoursformed by human cancer cells. Now, we use two mouse melanoma cell lines:B16F0 and B16F10 to perform addition mAbs treatments. Both cell linesform multiple metastatic tumours rapidly in mice. For untreated micecarrying B16F0 cancer cells that express endogenous PRL-3 protein (FIG.11A), metastatic tumours are found in adrenal (arrows indicated),livers, bones and abdomen (FIG. 11B, R-side). Again, we showed that thechimeric mAb could efficiency wipe out metastatic tumours formation inmany tissues of treated mice (FIG. 11B, L-side). In a parallel controlexperiment, dozens of metastatic tumours are found in lungs of untreatedor treated mice that carrying B16F10 cancer cells that do not expressendogenous PRL-3 protein (FIG. 11A), as one can see that there are nosignificant differences in numbers of lung metastatic tumours betweentreated (FIG. 11 upper panel) and untreated mice (lower panel). So far,the results obtained from PRL-3 chimeric mAb treatment on several PRL-3positive and negative cancer cell lines suggest that the efficiency ofthe treatment is tightly correlated with whether the formation ofmetastatic tumour is caused by the PRL-3-overexpression. If themetastatic property of cancer cells is not due to PRL-3 overexpression(B16F10 cells), the administration of PRL-3 chimeric mAb has no effectin blocking tumour formation (FIG. 11C).

Example 25 Discussion (Examples 15 to 24)

Most cancer patients die from metastases and not from their primarydisease. Cancer metastasis is a multistep process. The first importantstep of cancer metastasis involves neoplastic epithelial cells losingcell-cell adhesion and gaining motility, which drive cancer cells todisassociate from the primary site of the tumour and to invade adjacenttissue. The second step requires cancer cells to acquire an ability toget into the blood circulation (intravasation) of the host. These twoinitial steps might take a long time to incubate and achieve. Here, weused an experimental metastasis assay (reference A 19) and started ourexperiment at the step of intravasation by directly injecting onemillion of cancer cells into blood circulation via the tail vein of mice(on day 1) which mimic the rest of metastatic process. We treated theanimal with PRL-3 chimeric mAb on day 3, following by twice weekly ofthe mAb administrations. Taken together from the outcomes of theprevious (reference A16) and current antibody treatments, our findingssuggest: 1. the process of cancer metastasis is a long and difficultjourney; starting with one million cancer cells (seed), by the end ofthe experiment; we observed about 100 metastatic lung tumours inuntreated mice and about 10-15 metastatic lung tumours in mice treatedwith PRL-specific mAbs (reference A16), the data reflect that only about0.01% of cancer cells are estimated to reach their final destinationsuccessfully. 2. In this study, again, we found fewer numbers ofmicro-tumour in lung sections from treated mice comparing with thosefrom untreated mice (FIG. 10A); suggesting that the mAb could act onreducing the number but not the size of tumours. 3. The efficiency ofthe treatment is also highly associated with what time we begin tointroduce the chimeric mAbs. If the treatment is not started earlyenough but delayed to 1-week after cancer cells inoculation, the resultswould not be as good as early treatment (day 3); these findingsimplicate that PRL-3 mAb might play a role in neutralizing anderadicating the PRL-3 cancer cells when they are still moving andwondering in the blood stream. Delayed treatment might allow cancercells to have a chance to pass through the blood vessel (extravasation)and land on distal organ for seeding; the mAb might have less chance toarrest the PRL-3 positive cancer cells beyond extravasation and seeding.This hypothesis is supported by the prolonged presence ofDLD-1-EGFP-PRL-3 cells in the circulation of untreated mice and clearreduction of these cells in mice received mAb treatment. 4. Mostimportantly, PRL-3 chimeric antibody could only effectively block theformation of metastatic tumours that derive from the cancer cellsexpressing endogenous PRL-3; but not cancer cells that do not expressendogenous PRL-3. Therefore, our PRL-3 mAb treatment is very specific toits own antigen. Notably, the mAb acts better through the blood streambut not through local response, we generated xenograft tumours byinjecting one million of PRL-3 cancer cells locally in the hip area ofthe nude mice, we then injected mAb to the similar areas twice weeklystarting on day 3, we found that the mAb has no effect in reducing thesize of local xenograft tumours (data not shown).

The detailed cellular and molecular mechanisms responsible for PRL-3 mAbto inhibit PRL-3 mediated metastatic tumour formation in theexperimental metastasis assay are currently unknown and need to bedefined by future study. The results that PRL-3 chimeric mAb couldablate metastatic tumours of PRL-3 expressing cancer cells in mice areencouraging and suggest a concept for other intracellular targets inclinic. Our study might open up new and enormous opportunities forantibody therapy using mAbs against intracellular oncogene products totreat various cancers and cancer metastases. As PRL-1, PRL-2 and PRL-3are overexpressed in various cancers; we would anticipate the widelyneeds of the PRL-chimeric mAbs that could be the forerunners of novelmedicine to combat various PRL intracellular phosphatses associatedtumours. We could select and treat some types of cancer patients (forexample: who suffer pancreatic cancer) that would relapse-recurrencewithin a short period of time when the primary cancer is firstdiagnosed. The differences between treated and untreated groups ofpatient would be able to reveal if the chimeric mAbs have effects or notwithin a short period of time.

Example 26 Anti-PRL3 Antibody 318 Binds to both Intracellular andExternalised or Secreted PRL3 Polypeptide

An experiment is conducted as follows:

1. Grow A2780, HCT116, B16F0, B16 F10 cells in 10 cm culture dish eachtill 80% confluent.

2. PBS wash for several times.

3. Change medium (8 ml) into FBS-free overnight.

4. *next day, harvest medium and spin 3K, discard the pellet, spin 14Kagain and keep the supernatant (*lyses cells from the dishes, check eachcell line for PRL-3 expression in FIG. 12A)

5. use GAPDH as a protein loading control for cell lysates (FIG. 12B)

6. Check the medium under microscopy to make sure there is no cell inthe medium.

7. Concentrated the medium and run SDS gel for secreted or externalizedPRL-3 in culture medium (FIG. 12C).

The results are shown in FIG. 12.

FIG. 12A. Western blot demonstrates that A2780, HCT116, and B16F0 arethree cancer cell lines that express endogenous PRL-3 protein, whileB16F10 is a PRL-3 non-expressing cancer cells.

FIG. 12B. GAPDH is used as a protein loading control.

FIG. 12C. Western blot analysis on four culture media that are harvestedfrom the four cancer cell lines. PRL-3 phosphatase is found to besecreted out from B16F0 cells into culture medium while PRL-3phosphatase was not found to be secreted out from the rest of the threecells, suggesting that the secretion of PRL-3 phosphatase could relateto cell type specific phenomena. The data suggest that in vivo, PRL-3can be secreted into the blood stream in cancer patients. Phosphatasesecretion has never been reported in our literature.

Example 27 Epitope Mapping of Anti-PRL1 and Anti-PRL3 Antibodies

Epitopes for each of the anti-PRL antibodies 269 and 318 are mapped asfollows.

First, we exclude most of the amino acids that are identical among allthree PRLs. Second, we select the differences in amino acid sequencesamong the three PRLs. Third, 28 peptides are specially designed forthese specific regions that might be able to distinguish from each otherfor the binding of their respective antibody to its own specificregions.

We specially designed 28 PRL-1, PRL-2, and PRL-3 specific polypeptidespots and ordered from Genemed Synthesis, Inc. (www.genemedsyn.com). The28 peptide spots are arranged as a map in Table E1 below that alsoindicates each polypeptide sequence corresponding to the spots to FIGS.13A and 13B.

TABLE E1 28 peptides tested for epitope binding against anti-PRL1 antibody and anti-PRL3 antibody.  Reference A B C D E f 1TYKNMR TLNKFI NKFIEE VCEATY DTTLVE KEGIHV 2 PSNQIV KDSNGH NGHRNN SYENMRTLNKFT NKFTEE 3 VCDATY DKAPVE KEGIHV PPNQIV RDTNGH SYRHMR 4 TLSTFISTFIED VCEVTY DKTPLE KDGITV KAKFYN 5 PPGKVV YNDPGS KDPHTH HTHKTR TheTable shows a map representing an arrangement for each dot. Thepolypeptide sequences corresponding to the dots in FIG. 13A and FIG. 13Bare shown. Table E1 discloses the sequences in column A as SEQ ID NOS13, 19, 25, 30, and 36, the sequences in column B as SEQ ID NOS 14, 20,44-45, and 37, the sequences in column C as SEQ ID NOS 15, 42, 18, 32,and 47, the sequences in column D as SEQ ID NOS 17, 22, 27, 34, and 39,the sequences in column E as SEQ ID NOS 41, 24, 28, and 46, and thesequences in column F as SEQ ID NOS 18, 43, 29, and 35, allrespectively, in order of appearance.

Protocol for PVDF Dot Blot Membrane

Blocking Membrane

1) Pre-wet membrane with 100% Methanol for a few seconds until itchanges from opaque white to a uniform translucent gray when it isthoroughly wet. 2) Incubate the membrane in water for 45 minutes toelute the methanol. 3) Block in 3% BSA overnight at 4° C. with shaking.

Immunostaining

1) Incubate either with PRL-1 (#269 in FIG. 13A) or with PRL-3 (#318 inFIG. 13B) antibodies overnight at 4° C. with shaking. 2) Wash with PBStween-20 four times, 10 minutes each. 3) Incubate with anti-mouse HRP1:1000 2 hours at room temperature with shaking. 4) Wash with PBSTween-20 four times, 10 minutes each.

ECL Developing

1) Incubate with detection agent 5 minutes at room temperature. 2) Drainexcess buffer, place in plastic/saran wrap. 3) Develop in dark room.

Results

The results are shown in FIG. 13A and FIG. 13B.

FIG. 13A shows the results of a Western blot analysis to map epitopesfor PRL-1 (clone #269) mAb. Two positive dots indicate that the PRL-1mAb preferentially binds to two polypeptides (1a, TYKNMR (SEQ ID NO:13); 1b, TLNKFI (SEQ ID NO: 14)).

FIG. 13B shows the results of Western blot analysis to map epitopes forPRL-3 (clone #318) mAb. The two dots indicate that the PRL-3 mAbpreferentially binds to two polypeptide (4f, KAKFYN (SEQ ID NO: 35); 5d,HTHKTR (SEQ ID NO: 39)).

The results show that PRL-1 mAb (clone#269) and PRL-3 mAb (clone#318)may bind to epitopes that are formed by non-linearized sequences.

REFERENCES

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Zeng Q, Si X, Horstmann H, Xu Y, Hong W, Pallen C J.    Prenylation-dependent association of protein-tyrosine phosphatases    PRL-1, -2, -3 with the plasma membrane and the early endosome. J    Biol Chem. 2000; 275:21444-52.-   19. Li J, Guo K, Koh V W C. et al. Generation of PRL-3 and PRL-1    specific monoclonal antibodies as potential diagnostic markers for    cancer metastases. Clin Cancer Res 2005; 11:2195-204-   20. Britta W, Johannes L P, Laura J V. Breast cancer metastasis:    markers and models. Nature Review Cancer 2005; 5:591-602.-   21. Cooper S. Reappraisal of serum starvation, the restriction    point, G0, and G1 phase arrest points. FASEB 2003; 17 333-40.-   A1. Darrell C. Bessette & Dexin Qiu & Catherine J. Pallen PRL PTPs:    mediators and markers of cancer progression Cancer Metastasis Rev    DOI 10.1007/s10555-008-9121-3-   A2. Rouleau C, Roy A, Martin T S. et al. 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Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

The invention claimed is:
 1. A method of treating a PRL-3 expressingcancer in an individual suffering or suspected to be suffering from saidcancer, the method comprising administering a therapeutically effectiveamount of an antibody selected from the group consisting of: (a) anantibody which binds to PRL-3 and comprises a heavy chain variableregion sequence comprising SEQ ID NO: 10 and a light chain variableregion sequence comprising SEQ ID NO: 12; (b) an antibody which binds toPRL-3 and comprises a heavy chain variable region comprising SEQ ID NO:6 and a light chain variable region sequence comprising SEQ ID NO: 8;and (c) an anti-PRL-3 antibody capable of binding to an epitope HTHKTR(SEQ ID NO: 39) and which is capable of binding to an intracellularPRL-3 polypeptide by being capable of crossing the plasma membrane; or apharmaceutical composition comprising such an antibody to theindividual.
 2. A method of treating a PRL-3 expressing cancer in anindividual suffering or suspected to be suffering from said cancer, themethod comprising diagnosing cancer in the individual and treating theindividual by the method according to claim
 1. 3. The method of claim 1,wherein the cancer is selected from the group consisting of: a PRL-3expressing cancer; a metastatic cancer; colorectal cancer; ovariancancer; breast cancer; liver cancer; pancreatic cancer; prostate cancer;gastric cancer; lung cancer; penis cancer; cervical cancer; braincancer; esophageal cancer; bladder carcinoma; kidney renal cellcarcinoma; ovary lymphoma and skin melanoma.
 4. The method of claim 1,wherein said cancer is metastatic cancer.
 5. The method of claim 2,wherein said cancer is metastatic cancer.
 6. A method of treating aPRL-3 expressing cancer or inhibiting metastasis for a cancer expressingPRL-3, the method comprising administering to an individual in need ofsuch treatment a pharmaceutical composition comprising an antibody thatbinds intracellular PRL-3,wherein said antibody is capable of binding toan epitope HTHKTR (SEQ ID NO: 39) and is capable of crossing the plasmamembrane of an unpermeabilized cell, whereby said cancer is treated ormetastasis of said cancer is inhibited.
 7. The method of claim 1,wherein the antibody is a humanized antibody capable of binding to anepitope HTHKTR (SEQ ID NO: 39).
 8. The method of claim 6, wherein theantibody is a humanized antibody.